ANAEROBIC BACTERIA IN THE
CLINICAL tHCR03IOLOGY LA30RJ\TOHY
by James Ira fiiller
A thesis submitted to the faculty of the University of Utah in partial fulfi'ilr:i::nt of th(~ t'equ'i\,,(Wf~nts for the degree of
in cal Technolopy
Co 11 egf.:! of Phal""milcy
Un i ve rs i ty of Ij-i:ah August 1974 COPY}"'i ght©James Ira f··1i 11er 1974 All Rights Reserved UNIVERSITY OF UTAH GRADUATE SCHOOL
SUPERVISORY COMMITTEE APPROVAL
of a thesis submitted by
James Ira Miller
I have read this thesis and have found it to be of satisfactory quality for a master's degree. D�� �� i::�({{:fe>;(1J:I'>Y1f) Chainnan, Supervisory Committee
I have read this thesis and have found it to be of satisfactory quality for a master's degree. I " , "/, � / ../ '7
Date
Member, Supervisory Committee
I have read this thesis and have found it to be of satisfactory quality for a master's . degree. \ ,�-.. ) /.....-�')' ,' , '/. A /,2 flun _��- . " L:ccli--) . IC-�J- C/(_ ' .1 Date v David B. Roll Ph.D. Member, Supervisory Committee UNIVERSITY OF UTAH GRADUATE SCHOOL
FINAL READING APPROVAL
To the Graduate Council of the University of Utah:
I have read the thesis of James Ira Mi 11 er in its final form and have found that (1) its fonnat, citations, and bibliographic style are consistent and acceptable; (2) its illustrative materials including figures, tables, and charts are in place; and (3) the final manuscript is satisfactory to the Supervisory Committee and is ready for submission to the Graduate School.
� 'fJ. W� 'm 0,----- J�es N. Wilfert, ��-- Member, Supervi!lOry Committee
Approved for the Major Department 1��'{;:tir(ASCP )
Chairman /Dean
Approved for the Graduate Council TABLE OF CONTENTS
LIST OF ILLUSTRATIONS vi • • • • · .. . • • • • • • • • • • • • II •
ABSTR/1.CT ...... (. • viii
I. nrrROf)UCTION • I ~ e I • • • • • • I • • I e I I I ~ e • ~ 1
II. LITERATURE REVIEil • • .. I • • • I • • I • • • • • I • • 6
A. Anaerobic Systems. I • • • • • • • • • • • • • • 41 • • 6
1. Biological reduction • • • e • • • .. • • • • • • • 6
2. Physical reduction .. • • • • • • • • • I • • ~ e I 8
3. Evacuation • • • • • • • • • " I • • • • • • • • • 8
4. Use of inert gases • • f • • • , • • • • • • " .. • 9 5. Catalytic ianition of hydrogen and res i dua 1 OXY~JE.~n ~ • • • • • • .. 10
6. Reduction by phosphorus • • • 41 • • • ., . 12
7. Reduction by iron compounds •• .. e ., 12 • III • • • . .
8. Reduction by alkaline pyro9allic acid. • " • t • • 13
9. Agent in medium •••• • • • I • •.• • • • • • • • 14 B. Maintenance of Anaerobiosis. • • ·.. · . . . .. · ... . 14
t·1edi a tl • • • • • • • • • .. . • • • • • • • • • I> • 16
D. Gas Chr-ona tO~l raphy • c • • • I • • • • • • • ~ • ~ • • 16 E. Relative Frequency of Anaerobes in C'linical fiateY'ial 17
III. MATEfUALS o f'lETHODS • • • • • I 27 • 41 • • • • • • • • • • • A. Sources of Specimens ••••••••••••••••• 27 28 B. Transport Hethods • • • • • • • • • • • • tl • • I • II • c. Isolation riethods ••• · ...... 31 1. Anaerobic glove box · ...... 31
2. Roll tube apparatus • • • • • • • • • • • • • • • • • • 35
D. Identification of Anaerobes • • • • • • • • • • • • • • • • 38 1. Gas chromatograph. • • • • • • • • • • • • • • • • •• 38
2. Media used ••••••••••••••••• • • • •• 42
a. Media for Isolation • • • • • • • • • • • • • • • • 42
b3 Media for Identification • • • • • • • • • • • • • 45 3. Identification procedul"es • • • • • • • • • • • • • •• 46 IV. RESULTS...... 52
A. Results of Gas Chromatography • • • • • • • • • • • • • 52
B. Culture Results ••••••• ." . " • • • • • • • • • • 65
V. DISCUSSIOn ••••• f •••••••• e ••••••••• 84
BIBLIOGRAPHY • • • • • • • • • • • • • • • • • • • • • • • • • 91
VITA •• , •••••••••• 0 ••••••••••••••• 96
v LIST OF ILLUSTRATIONS
TABLES
I. Methods for obtaining anaerobiosis •• • • • • • • • • • 7
I I • r·1os t common 1y encountered anaerobes • • • • • • • • • • 19 III. Frequency of co-isolation of anaerobic species with aerobes and other anaerobes • • • • • • • • 20 IV. Anaerobic organisms in infections in predisposed patients from literature survey • • • • • • 21 V. Anaerobic organisms in infections in pred is posed patients. Hads\'Jorth VAH-UCLA • • • • • • • 22 VI. Summary of anaerobic bacteria isolated
according to strain and source ••• • • • • • • • • il 24 VII. Incidence of various anaerobes as normal flora in humans • Q ~ •••• ~ 0 ••• • • • • • • • • 25
VIII. Anaerobic bacteria fran human infections ~ost frequent ly 5 ubmi tted to the CDC anaet~obe 1abQra tOl"Y • • 26
IX. Anaerobes and aerobes recovered " . . . . (, ...... 77 x. A. Aerobes isolated from vario~s sources •••• • • • 78 B. Anaerobes isolated from various sources 79 • • • • II •
XI. Anaerobes and aerobes isolated concurrently II • • • • • 81
XII. Anaerobes isolated concurrently with various sources • • 82
XIII. The most frequent anaerobic isolates • • • II • • • • • • 83
GRAPHS
frCtqilis 55 fraqilis. Volatile fatty acids 54 lA 11.. , ... • • • • •
lB fraqilis 5S fraqiliso r,1ethy1ated derivatives 55 B. «( .1fII • • • • 2A B.. --fraoilis S5 fraqi 1i~. Volatile fatty acids • • • • • 56 28 B. fraoilis S5 -fraa;lis., .. Iliethylated derivatives • • • • 57
3 Bf; melaninoqen;cus. Volatile fatty acids I' 58 '...... ,.~ • • • • • •
4A F. nucleatun1. Volatile fatty acids • • • • • • • • fl • 59
4B F. nucleatuHl .. r·1ethyl a.ted derivatives • • • • • • • • • 60
5A F. necroEh~. Volatile fatty acids • • • • • • • • • 61
5[3 F. necrophorum. Volatile fatty acids • • • • • • • • • 62
6A V. alcalescens. Volatile fatty acids • • • • • • • • • 63
6B 'i. alcalescens. i;lethyl ated derivatives • • • • • • II • 64
7A Lactobaci lli& ~. Volatile fatty acids • • • • • • • • 66
78 La cto9.. a ci 11 u.~ .::..E.. f,1ethyl ated derivatives • II • • • • • 67
8A Lactobaci 1l.u.~ .22.. Volatile fatty acids • • co • • • • • 68 8B Lactobacillus -SDc f:1ethylated derivatives • • • • • • • 69
p& I' 9 ~9 Volatile fatty acids • • .. • • • • • • • · 70
10 histolvticun. Volatile fatty acids co 71 £. ~~1lt;~(~\~..;....:.i ••""'Z • • • • • • • •
11 £. d i f.fis: i 1e,. Volatile fatty acids • • • .. • • • I' • .. 72
12 nqen~, Volatile fatty acids t t 73 C. Ecrfri • • • • " • · 13 f_e~tostrGptococcus anacx.. oJd us. Volatile fatty acids · .' 74
14 PeptocoC':.,.ct§. r.laan..u...:~! Volatile fatty acids • • • • • II • 75
15 Pepto(;:9_ccu,s .2!evoti i • Volatile fatty acids • • • II • • 76
PHOTOGRAPHS
I. Anaerobic glove box used at the University of Utah. • • 34
I I • Roll tube apparatus used at the University of Utah • • • 37 FIGURE
I. Form used for recordin~ rcsults.e •••••••• t.f ••••••••••••••• Sl
v;i ABSTRACT
Recent advances in anaerobic microbiolo~y require more sophisticated techniques and nev/er knm'lledge. Using these ne\",er methods gives results that have not been available in routine clinical laboratories before •. Interpreting the data is made difficult because of the lack of experience of most medical technologists. Many questions come up such as: Hhat is the frequency of anaerobes? Hm'I often do the vari ous spec; es such as Eubactcri urn jent,u:n., occur? tihat is the frequency of co-isolation "lith aerobes and othc r anaerobes? A rev i ei'} of the 'I i tera tu re is of no t"ea 1 he 1p because of the f,lany different methods that have been used for isolation and identification. Another reason is that ~uch of the pub 1; shed "/ork has been don e in research or reference 1aboratori es and do not necessarily relate to the situation in a routine clinical 1aborator·y. The most recent methods developed for anaerobic work include an anaerobic glove box and the roll tube arraratus. Both of these methods were used to determine the frequency of isolation of the various anaerobes in clinical specimens submitted for anaerobic culture to the Clinical Labor'atory at the University of Utah f'iedical Center. Identification procedures included the use of a thermal conductivity gas chromatograph. A total of 114 specimens Here processed. 75.4~& contained at least one anaerobe. The most co~mon specimens were from abdominal sources (44%). 81.6% of the specimens contained either aerobes or anaerobes or a combination. Of the cultUl"e positive specimens, 92.5% had anaerobes and 64.5% had aerobes.
Bacteroides fragili~was mixed most often with aerobic organisms
(13~;). Of those mixed cultures 10/21 {48~~} Viera \vith Esche.dch~ coli. Both are gram negative rods. The anaerobic gram positive cocci were in mi xed cul ture most often \"; th the aerobi c gram pos; ti ve cocci (42%). The most frequent anaerobic isolates were: o. fragilis 18%
PSt magnus 8%
Fusobacterium sp. 8~; Eubacterium sp. 7%
Bacteroides srI! 7%
PSt intermedius 6% Ps. anaerobius 6% B. oralis 5%
c P. aenes 510/ Bifidobactcr;um 5%
Veillonella sp. 5~170 Pc. assacharolyticu5 5% Pc. prevotii 5% These data indicate that unless a primary plating method is used that \'Iil1 alloH for separation of both aerobes and anaerobes there will be a tendency to overlook anaerobes because of the frequency of
ix co-isolation vlith aerobic organisms that have the same general gram morphology. The same reasoninn holds true for the gr'am stain of the original specimen. If a gram negative rod was seen on the original smear and I. coli \'/aS isolated on the aerobic plates the assumption that all organisms present in the specimen were isolated does not al\'lays hold true.
x I
INTRODUCTION
Up until the last fevl years, the technology of anaerob'ic bacteriology has been far behind the procedures used for most other groups of organisms. It has been said that anaerobic bacteriology is at the present time v/here routine bacteri ology \Vas about the turn of the century. Presently most laboratories are unable to isolate any but the mos t common of the anaerobes sud1 as Cal os tri di hLril
££rfri n!1en~, anaerobi c streptococci and bacteroi des. The finitions aerobic, anaerobic and fucultativc anaerobic are arbitrary and difficult to define, Ther'e is a Hide range of or'gani sms in re 1at; on to thei r oxygen requi rements. Some organi SillS vri 11 not grOi'1 ; n an atmosphere of reduced oxygen \t/h; 1e other's vJi 11 not tolerate exposure for more than a few minutes at a time. Most organ; srns can be grouped accord; ng to the i r oxygen requi rCP1ents. The following are groups of organisms based on their requirements for oxygen or their sensitivity to it. Groups four to eight are presently considered to be the anaerobes.
1. The organ i SillS in thi s group requi rc oxygen to groH. The; r
grm·!th is directly proportional to the amount of oxygen
present. These are the stri ct aerobes such as pseudor.1ona~.
.§..E.:.. and flo; ssg.ri a ~ Any decrease in the oxygen concentra
ti on t,/i 11 decrease the gt"oVlth rate. 2
2. These organ; sms \'Ji 11 gro\,1 in the presence or absence of oxygen but will grow better with it prescnt. Most of the organisms encountered in clinical specimens at the present time belong in this classification. They are knmlJn as the facultative anaerobic organisms. 3. This group of organisms grow equally well aerobically as well as anaerobi cally. These are a 1so knot-In as facul tat; ve anaerobes. 4. The fourth group of organisms is hard to label as anaerobes or aerobes since they will not grow aerobically but will not groVi anaerobically either. They \"i 11 vJithstand oxygen in concentrations up to ten percent. Organisms of the genus
C~mpyl o~.£!: \'Joul d be put into thi s group. 5, Organisms that grow aerobically but grow much hetter
anaerobically are put into this category. An example of t~li5
type of organism is Clost,r~... 9um histoly.ticum.· 6. This group of organisms will not grow aerobically but are not very sensitive to molecular oxygen and can survive for some
time exposed to air. This group includes propionibuct~riu.m. acnc1., an anaerobe found on the normal skin of most people. 7. Strict anaerobes that a}"e oxygen labile and \'Ii11 not survive
prolonged exposure to oxygen are in this group~ OrganiSMS such as Bacteroides ir'aai,.1is. Fusobacterium sp.· and many other anaerobes commonly found in the clinical bacteriology
1abora tory e 3 B. The last group includes the extl"ernely oxygen sensitive anaerobes that \'Ii 11 not tolerate exposure to oxygen for even short periods of time. These organisms have not yet been proven to be of any clinical significance and will not be discussed in this paper. Until recently it was considered adequate to identify the anaerobes as one of the several large groups: 1. "Bacteroi des ". Any anaerobe that vias a gram negati ve rod was placed here. 2. "Anaerobic strep". All anaerobic gram positive cocci \'Jere grouped under this heading. 3. ,el as tri d,; ,u!TI ,E.erfri nil§ns has been s peei a ted because of the serious nature of the disease that it causes and because it is relatively easy to identify. 4. All other Clostridia were not speciated but labeled as
Cl o~tri d.1.!.!B, ~... not l!.£.rfi"',i.t~g~.
This way of identifying organisms is comparable to ca1lin~1 all .gram negative r"ods from the stool as Enterobacteri.aQceae. There are differen ces in pathogenicity and sensitivity patter'ns of the organisms in both grours~ At the present time, there are many 1aboratories that use only thioglycol1rlte broth for all of their anaerobic culturing. If there is a mixed culture, or the anaerobe does not gro\'J vJe11 in thicglycollate, the chances of isolating it are considered to be very poor. Since many laboratories do not possess anaerobe jars, they cannot plate out the specimen or subculture the thioglycol1ate broth to obtain pure cultures 4 to enable identification of the organisms. Of those that have anaerobe jars, few use them to plate out their speciDens directly on a routine basis. Several investigators have estimated that the average laboratory isolates only about five to ten percent of the anaerobes present in routine clinical speciMens. In 1908. Runebert (Smith. Holdeman 1968) made a statement that caul d have been made today, "One of the rna; n reasons v/hy our knowl edge about the anaerobic bacteria, .especially in human pathology, is not on the same plane with the knowledge of the' living habits and importance of oxygen tolerant bacteri a comes fl"om the techni ca 1 di ffi cul ti es the pure culture and close study of the living habits of these bacteria always have given. Extensions of knO\>Jledge of anaer'obic bacteria have usually gone hand in hand with improvements of technical procedures in culture, and still do 50," Since the early 19005, many laboratories have been working on the problems of isolation and identification of anaerobes. As newer techniques are developed they are becoming more practical for routine use in the clinical laboratories. Since these organisms are unfamiliar to most bacteriologists, many questions have been raised concerning which anaerobes are expected in any given speciPlcn and \~hat their relative frequencies are. For example, what is the most common anaerobic organism found in pet\itoneal abscesses? Is an anaer'obe such as 1.~i\C_~£!.1.u!!llentul~ very common in this type of specimen? The purpose of this study \'Ia5 to attempt to answer these questions by developing a practical anaerobic system or systems and selecting speciMens from those submitted to the clinical laboratori[;s of the University of Utah Hospital to recover the 5 anaerobes and identify them as far as practical and necessary. Isolation procedures included the use of a glove box and a roll tube apparatus as vlell as the common anaerobe jars. Identification proce dures were carried out with the help of a gas chromatograph to identify the vo1atile fatty acids produced by these organisms. II
LITERATURE REVIEH
A. Anaerobi £ .. systerns" Hall, in 1929 (I-tall, 1929) publ-ished an extensive review of the principles used in the cultivation of obligate anaerobes. In this review he discussed methods of obtaining and maintaining anaer'obic
~onditions and pointed out that as a rule. a combination of one or more of the basic principles ViaS involved. It is very One of the s ir;lP 1es t m{~thods foY' removi ng oxygen fr-om the grovJth chamber is to U5C an organism that uti lizes CXY~len such as Stap,h:ylo c9.c.c.us .CiUr.~t~, or Scr'rnti ~ ~ce~~ ( SJc; e ty of Ameri can Bacteri 010- gists, 1957). The technique involves the grovJth of the aerobe on a separate r~,ediur:1 from that \'Jhich the anaerobe is to be grown. The tV/O plates arE! then sealed t0gether "lith tape placing the media facing 7 TABLE I r1ethods for' Obtaining and f1aintaining Anaerobiosis (Hall 1929) 1. Reduction of oxygen tension A. ~iolpgical reduction Aerobe-anaerobic symbiosis Symbiont in medium Symbiont in air chamber Use of animal and plant tissues B. Physical.Is reduction Boiling Evacuation Use of inert gases Agent in air chamber Catalytic ignition of hydrogen and residual oxygen Reduct'j on by phosphorus Reduction by iron compounds Reduction by alkaline-pyrogallol "nen tin ned; Uf'l 2. t1a'intenance of reduced oxygen tension Deep medium seals Insoluble liquid seals B. !\; r c11mnber' ,sealGd Fusion of glass outlet Mechanical seals 8 each other and forming an airtight chamher with the two petri dish bottoms. As the aerobe grov/s it reduces the envi ronment ins i de the chamber allm'ling the anaerobe to initiate growth. A method using vegetable tissue was described in 1935 (McClung. r~cCoy and Fred, 1935) in \'Jhich oats. grain or other tissues, particularly chopped Irish potatoes. are used to remove oxygen from the air. The method is very simple, easy to perfol"'m and does not require any elaborate materials, Oats are placed in the chamber to fi 11 about one tenth of the capaci ty of the jar and moi stened \.Ji th tap \l/ater. Plates to be cultured are added and the container sealed with p1asticene or similar material. The moistened oats remove enough of the oxygen in the atmosphere to all 0\,1 anaerobes to gro\,l. The time ..'equired to obtain anaerobic conditions is quite long and only the more oxygen tolerant or'ganisms or spore formers \'/ou As a liquid is boiled, the dissolved gases are driven off. This is the principle involved in removing oxygen from media by boiling. 3. Evacuation, ..." t m This procedure involves the removal of much of the original air by vacuum and rep 1ac; ng it \I/i th an oxygen free gas. The evacua ti on and repl acement are repeated three times. Thi s procedure has been adapted to be used with the cold catalyst (BBL, Engelhard Ind •• Torsion Balance Co.). The jar is evacuated and flushed three times with a gas 9 contai ni ng approx; mately ten percent hydrogen to react \'1; th the oxygen in the presence of the catalyst. This removes the last traces of oxygen that are not removed by evacuation and that may be liberated from the media or that may be accidentally introduced into the system. 4. Use of inert gases. This method ViaS first descri ted many years ago (Hall, 1929).. In 1950, Hungate (Hungate. 1950) described a method of using inert gases to flush out individual tubes to provide an anaerobic atmosphere inside each tube. He used a bent pas teur p'i pette to ; ntl"oduce the gas into the tube and 5 toppered it \'Ii th a rubber s topper as the cap; 11 ary pipette vIas vJithdra\'ln. No allowance VJas made for residual oxygen; since, theoretically. no oxygen is left after flushing because this method uses Pre-Reduced Anaerobically Sterilized media (PRAS), and therefore has no dissolved oxygen in it. This method ho.s been given the liamc "roll tube procedure" because to prepare an agar surface ; ns; de the tUbes it is necessary to roll the tubes containing melted agar horizontally until the agar has hardened on the inner surface of the tube. Moore (t~ool"e. 1966) further developed the roll tube method and devised a complete system to enab1e one to inoculate ttH~ roll tubes with relative ease and to readily inoculate the biochemicals necessary for identification (Be11co Glass Co., Vineland, N.J.). Thi s ; s the method us ad at the an aerobe 1aborator'y at the Vi rg in i a Polytechnic Institute at Blacksburg, Virginia (Holdeman. 1972). 10 5. Catalxtic iqni~ion of hy.qrogen and residual oxygen. laidlaw (Laidlaw. 1915) described a combustion principle for obtaining anaerobiosis using a platinized carbon catalyst. The first uses of this involved placing the catalyst on a wire in a cork and heating it before placing in the tube of hydrogen. A hydrogen generator was used to fill an upside down tube with hydrogen. The tube had to have enough hydrogen in it to prevent an explosion when the hot cata lys t \Alas i ntraduced. The reacti on of the hydrogen \t/i th the oxygen produced enough heat to keep the reaction going until all of the oxygen \,/as reacted. t>Jhen us i ng 1a l"'ger con ta i ners tit vIas very important to be sure that the bacteriological loop was cooled suffi ciently to avoid an explosion with the unreacted Ilydrogen remaining in the container, t·klntosh (tklntosh and Fildes. 1916, and rkIntosh and Fildes. 1921) later used the Laidlaw principle to devise an anaerobe jar. The catalyst had to be heated just prior to introduction into the jar and explosions v/ere not unCOMmon. Smillie (Smillie, 1971) refer'red to the anaerobe jar of !1clntosh as the "r·1clntosh bomb II • He changed the jar in t\'IO significant waYSt The first \'Jas the introduction of the hydrogen into the jar after it \las closed. The hydrogen passed over the catalyst as it entered the jar and therefore did not build up high enough concentrations to permit an explosion. The second change was that of.using electricity to heat the catalyst. This \'Ias a big step in that the catalyst could be reheated later to remove any traces of oxygen that \'/ere dissolved in the media or left over after the first reaction. It is interesting to note that Smillie sa-id, IIBy this technique the organisms of poliomyelitis gro\'J upon ascitic agar slants." 11 An improved anaerobe jar used the principle of the Davey Miners safety lamp (BrOt-Jn, 1921; Brot-tn, 1922). The catalyst and a coil of nichrome wire used to heat the catalyst were enclosed in a copper wire screen. This jar was further improved by Brewer (Brewer, 1939). This improved jar has been used for many years. The electrical wires used to heat the catalyst were changed so that they did not pass into the jar. This was done to prevent accidental explosions due to short circuits in the heating \'Jires. The BreVler jar is essentially the same as the Brown jar. Evans (Evans, 1948) modified the Brewer jar so that the gas replacement technique could be used vlith it. The relatively recent development of a cold catalyst of pa1adinized alumina (Torsion Balance Co., Engelhard Ind. II BBL) has brol1!)ht about another revolution in the use of anaerobe ja.rs. This catalyst requires no heating or outside source of energy which makes it applicable to field usc and can be used in the smallest of laboratories. It does not produce enough heat to ignite the hydrogen air mixture, so there is no danger of explosions as there was with the older jars. This is the system used in -most anaerobe jars available today. Brewer (Bre~er, 1966) developed a self-contained carbon dioxide) hydrogen generator envelope for use in anaer'obe jars equipped \·Jith the cold catalyst. It cons'ists of a hydrogen producing sodium borohydride tablet and a carbon dioxide producing tablet of citric acid and sodium bicarbonate. The carbon dioxide was included because some of the anaerobes require it for gro~th (Rose. 1942; Wilson, 1964). This envelope has eliMinated the need for regulators and tanks of gas or 12 other sources of hydrogen. To use the system, ten milliliters of tap water is added to the envelope and placed in the anaerobe jar. The cold catalyst reacts the hydrogen and the oxygen together to produce water, which condenses on the sides of the jar. Enough hydrogen is , produced so that any residual oxygen, or oxygen that is accidentally introduced in small amounts, will be immediately taken care of by the catalyst and the jar remains completely anaerobic until opened. 6, Reduct; on by phOSDho.. ~~ .. ~. This systerrl VJas used in California until recently (Lynn Greer, Salt Lake City, Utah. personal communication). It removes sufficient oxygen for mas t anaer'obes and does not requi re e1 abo rate apparatus. Yellow or white phosphorus is placed in a small container inside the chamber and the chamber sealed. The phosphorus is stored under water and as the phosphorus dries it ignites spontaneously and burns as long a's there is oxygen present. I f the phosphorus does not i gn; te spon ta neously but gives off a grey smoke, it must be ignited with ~ match before the jar is sealed. A small amount of tap water is place~ in the bottom of the jar to remove the P205 ~/h;ch forms. Using this method accidental burns are not uncommon and are very painful (Society of !\me,-';can Dacteriologists, 1957). This Method should b(~ used only be competent personnel and is not a good system for routine use in most laboratories. 7 • 8,c;.9.. l!.S:,t i on hi i ron compounds II The use of iron compounds for reduction depends on the maintenance of a large surface area of iron ;n contact with the atmosphere. Parker (Parker, 1955) used iron wool treated with acidified copper sulfate 13 solution to remove oxygen. Co~paring this method and the McIntosh and Fildes jar. no significant differences \l/ere obtained in the bacterial counts by the tV/o techn; ques. HovJever, the rklntosh and Fi 1des jar frequently failed due to small air leaks, while the iron wool jars gave consistent and effective results. Attebery (Attebery, 1970) adapted the method of Parket for use in miniature jars (35mm fi 1m containers). These "rnini" jars vlere compared to Brewer jars and gassed out "roll tubes and found to be equally effective in keepinq Bacteroides melanino~enicus and Fusobacterium ..... ~r 7'T ...... fusiform~ v'iable for six hours in inoculated tissue. This system \'Jas also used to cultivate obligate anaerobes on agar slants. Portions of Alka-Seltzer tablets were the source of carbon dioxide. 8. " Reducti on bv a 1ka 1 i no nyrooa 11 i c aei d. 'Ii. r"'-"~ I\nother chern; ca 1 method for remo'l; ng oxygen i 5 the QXY!1en abSOl"pti ve capac; ty of the rt;acti on betvlOcn a 1 ka.l i clnd pyrogn 11 i c ac; de The Spray (SpraYt 1930) dish is used fer plate cultures and used the above reaction. It consists of a glass petri dish top and a special bottom which al10V/5 the regular petro; bottom to fit into it at the top. It is fair'ly deep and has a raised ridge across the center to keep the t\,IO so1utions separ-ate until the dish is closed and tilted, a11m'/ing the two solutions to mix together. This reaction may be used in a test tube. The inoculated tube has a plug of cotton placed below the lip allm'Jing room for' the stopper. The pyrogallic acid is placed on the cotton and then the sodium hydroxide. The tube is quickly stoppered and inverted to pY'cvent the reaction mixture from running over the inoculated slant .. 14 9. Anent i n n~di ur~. The Brewer anaerobic culture dish (Brewer, 1942) is a single plating device that does not require any added equipment. This dish is used with media containing strong reducing agents. After the regular petri dish is inoculated, the top is replaced by the Brevler anaerobic lid. The lid ;s designed so that the periphery touches the surface of the agar. The center is raised forming a small air pocket. The entrapped oxygen is removed by the reducing agents in the medium. Metallic iron was included in liquid media as a means of reducing the media to promote the groVlth of anaerobes by Spray (Spray. 1936) and HaYHa BII !" f in tenance of an acrobi os i ..~ .. The method used to maintain anaerobic conditions depends on hoVl those conditions were obtained. If a tube of media was boiled, the only thing that needs to be sealed is the Medium and a layer of vaspar or mineral oil over the exposed surface will maintain" adequate anaerobic conditions. A mechanical seal could be used also t such as a rubber stopper or sealing a tube by fusion of the glass outlet. Anaerobe jars were originally sealed with plasticcne (Hall. 1929) or other material. but the prcsentdday anaerobe jars have a rubber ring that is used to seal the chamber. This rina is used over and over and eliminates the problem of getting a good seal using a tlrope" of plasticene. These jars are made of stainless steel (Torsion Balance Co.) or a durable rigid plastic (Baltimore Biological Laboratories). Recently a disposable anaerobic system was developed using a trip"le laminated plastic bag in combination v!ith the cold 15 catalyst and the Gas Pak envelope (BBL. This plastic bag is impermeable to oxygen) (Dov/ell, CDC. Atlanta. Georgia, personal communication). Another significant change in anaerobic methodology came with the introduction of the glove box. Rosebury (Rosebury, 1964) developed a box that was made of stainless steel and had a set of gloves on the side to permit the operator to work inside while maintaining the cultures under anaerobic conditions. Samples to be cultured and materials to work with were introduced into the chamber by means of an "interchange lock". This lock had b'IO doors, one on the inside of the chamber and one on the outsi de. After the specimens \'JCl"e placed in the lock, using the outer door, the lock was then evacuated and fi 11 ed \'Jl th an oxygen fl"ce gas. The inner door ViaS opened an d the specimens introduced into the chamber. For volatile materials, the i ock iI/aS evacua ted at a lower va cuum an d fl us hed \'1; til oxygen free gas three tiroos to prevent boiling or the agar from corning out of the bottom of the petri plates. Aranki (Aranki. 1969) made a qlove box that was made of clear polished vinyl and therefore much less expensive. The entire box \vas made of thi 5 1l1ateri alto all 0'.'1 unobs tructed vi si on in any prtrt of the chamber. Because this plastic is slight1y permeable to oxygen, the cold catalyst was used inside with a fan to circulate the inside atmosphere over it. The gas mi xture used \lJas 80% nitrogen t 10% hydrogen and 10% carbon dioxide. Similar' chambers are available commercially (Coy r'1fg. t (\nn Arbor, r1ichigan). 16 c. ~. Pre-Reduced Anaerobically Sterilized (PRAS) media has been reduced previous to sterilization and sterilized under anaerobic conditions. The media never comes in contact with the air after being hydrated and reduced. Reduced medi a on the other hand has been prepared ; n the usual manner and then placed in an anaerobic atmosphere to reduce it pr; or to use. f\1any 1aboratori es recommend the use of reduced medi a (Martin. 1971) or PPJ,S media (Ho1deman t 1972; f,1arymont. 1972). ~'Jhen pre-reduced media instead of standard media are used, the isolation of anaerobes vlill dramatically incr(~ase {Harymont, 1972; r1oor'e~ 1969}. rl1cf4i nn (ritctili nn, 1970) campa red routine Cll 1tur'i ng medi a and PRl\S medi a and concluded that an increase of more than 50% \'/as possible using the reduced media. Some laboratories feel that if fresh media are used and not reduced YOLl can recover all of the clinically significant anaerobes (Rosenblatt. 1972). De Gas .s!lromat99l"aphy. In 1941 ilartin (f~artin and Synge. 1941) made the suggestion that the mobile phase of chromatography could be almost anything including gas. It wasn't until ten years later that the first working gas chromatogroph \'las developed' (James and r1ar'tin, 1952). The fil"St detectors were quite crude by present~day standards. One of the first was simply a tube of indicator solution in which was dipped a capillary tube that Has attached to the end of the column (Porter, 1969). The first real detector was the gas density balance (James and l'~artin I 1956). 17 Gas chromatography has found several applications in the field of microbio109Y. J\mong the more significant developments has been the identification of certain bacteria (Brookes, 1971; Brookes, 1970; Ch a r 1eSt 1963 ; Ell end e r s 1970 ; Hen; 5 t 1966; t·, i t r ukat 1970; 1·100 re t 1966). Most methods used for anaerobes detect the volatile fatty acids produced such as acetic. butyric, and propionic acids. Gas chromatog raphy is presently being used in most anaerobe laboratories for help ih identification. The detectors that are most commonly used are the thermal conductivity (Holdeman, 1972) and the flame ionization (Sutter, 1972). Relative frequencv of anaerobes in c1iriica1 material. E. "... The frequency of isolation of any given group of organisms depends on the way they are isolated. If the method used does not allow for the isolation of some of the orqanisms the frequency of that group of organisms will be lower thon reported by another system that is ahle to recover them regularly. Hith so many different "Jays of obtaining and maintaining anaerobic conditions. it would seem that there would be, different frequencies of the various ·anaerobes reported. Because of these differences, on]y the recent literature will yield reliable inf'orrnation conccr-rring the relative frequencies of the anaerobic microorganisms. Moore (Moore, 1969) using the roll tube procedure and PRAS media reported that in a study of 300 specimens of lung, urogcn'ita1 tract. brain, blood and other sites over 85% contained obligate anaerobes. Most of these specimens contained more than one species of anaerobes. As many as seven different I~inds of anaerobes were isolated from 18 some infections. In the same study. the incidence of species of anaerobes isolated from 81 consecutive clinical specimens was reported. These organi Sr.lS are shov/n in Tab 1e I I. In 1970 (Cato, 1970) it was reported that 78% of the clinical abscesses in human or animal organs or tissues contained anaerobes in pure or mixed culture. The number of strains tested for each of the anaerobes is reported. However, because this is a reference laboratory, the number of strains studied is only an indication of the frequency and not the true frequency. Plating the specimens directly on enriched and selective media and ~sing the Gas Pak (SBL) anaerobic system. anaerohes were isolated in almost 40% of all tile bacteriologically positive specimens (Zabransky, 1970). This represents slightly more than otlG-rluarter of all specimens examined. The frequency of co-'isolation of anaerobic species \'Jith aerobes and othei" anaerobes vIas reported and shov/t1 in Tab 1G I I I. In a literature survey of 306 predispcsed patients, the anaerobic organisms reported by other authors and the number of isolates of each of the anaerobes is given in Table IV (Finegold, Marsh, Bartlett, 1971). The most co~non anaerobe was B. fraqilis with 96 isolates. .... l1 ~ In the same article, the expel"'ience at \,Jads\'JOrth Hospital in Los Ange 1es, Ca 1i forn; a. \AJaS presented. The next most common group \'-Jas the unidentified gram negative rods "lith 60. Clostridi_um. ~icum Ni th 27 i so 1ates vias ; so 1ated more frequently than f.. E,erfri n~en5 which had 26 isolates. The anaerobic organisms in infections in 68 predisposed patients is presented in Table V. 19 TABLE II Most Con~on1y Encountered Anaerobes (Moore. 1969) ORGANISnS % Bacteroides fragilis 17.4 Bacteroides species 13.9 Clostridium perfringcns 9.0 Eubacterium lentum 5.6 Clostridium species 5.6 Peptostreptococcus magnus 5.6 PeptostreptocoCCU5 species 5.6 FusobacteriuM species 2.8 Catenabacterium catenaforme 2.8 Catenabacterium filamentosum 2.8 Peptostreptococcus anaerobius 2.8 Peptostreptococcus interMedius 2.8 20 TABLE III Frequency of Co-isolation of Anaerobic Species \Vith Aerobes and Other Anaerobes (Zabransky, 1970) With aerobes In pure With other With and other culture anaerobes aerobes anaerobes Strains SPECIES No. No. % No. % No. % No. % Bacteroides fragilis 150 31 21 41 27 36 24 42 28 Bacteroides funduliformis 26 4 6 23 4 15 15 58 Bacteroides (other) 51 8 16 12 24 8 15 23 45 Fusobacteriunl spp. 36 0 0 14 39 6 17 16 44 PeptostreptocoCCllS spp. 67 9 13 14 21 19 28 25 37 Peptococcusspp. 71 1 ] 16 12 17 29 41 19 27 Clostriduim perfringens 41 4 10 10 24 6 15 21 51 Clostridium (other) 20 0 0 5 25 3 15 12 60 CorynebacteriUlTI (ani:lerobic) 20 5 25 3 15 7 35 5 25 21 TABLE IV Anaerobic Organisms in Infections in Predisposed Patients From Literature Survey (306 Patients) (Finegold. f1arsh, Bartlett. 1971) Uumber of cases One anaerobe isolated in pure culture 251 T\,lo or more anile robes ; no facul tat; yes 26 Both anaerobes and facultatives isolated 29 Anaerobes Isolated Number of isolates Unidentified anaerobic GNR 60 Bacteroides fra0ilis 96 Bacteroi des Ol"ll 1; s 7 Bactel"oi des tri elloi des 2 Bacteroides Mclaninogenicus 1 Bacteroides clostridiiformis 1 Bacteroides variegatus 1 NCDC group F-l 3 Fusobacterium species 8 Fusobacterium fusiforme 9 uFusospiroclieta"1 O1"Oanisr:ls" 8 Fusaei 11 us qi rans 2 ~phaerophorus species 1 Sphaerophorus necrophorus 18 Sphaerophorus varius 1 Dialister 1 Anaerobic cocci 8 Anaerobic streptococci 24 Microaerophilis streptococci 5 Clostridium pcrfringens 26 Clostridium septicum 27 Clostridium butyricuM 1 Clostridiun paraputrificum 1 Clostridium oedcmatiens 1 Clostridium sphenoides 1 Clostridium species 23 Eubacteri um 1entum 1 Corynebactcri urn acnes 1 Actinomyces spec; es 1 22 TABLE V Anaerobic Organisms in Infections in Predisposed Patients ~Jads\'lOrth VAH .. UCLA Data (69 epi sades in 68 pati ents) (Finegold, Marsh. Bartlett, 1971) Humber of cases One anaerobe isolated in pure culture 26 Two or more anaerobes isolated; no facultatives 14 Both anaerobes and facultatives isoalted 29 Anaerobes Isolated Number of isolates Bacteroides fragi1is 45 B. melaninooenicus 8 B. oral;s .. 2 B. corrodcns 1 Fusobacterium fusiforme or nucleatum 6 Sphaerophorus varius 1 Sphaerophorus species 3 Unidentified anaerobic GNR 7 Anaerobic cocci 6 Anaerobic streptococci 10 t'ii croaerophi 1i c cocci 3 11; croacrophi 1; c streptococci 16 Clostridium Dcrfrin0cns 3 Clostridium innocuum 1 Clostridium species 5 ActinoMyces israelii 1 Bifidobacteriur'l species 1 Corynebacter"; um acnes 1 Lactobacillus 4 23 Using a constant flow carbon dioxide cabinet to store reduced media for plating and a constant flovl anaerobic jar assembly to hold inoculated media until placed in an anaerobe jar with a Gas Pak (BBL). Martin (Martin, 1971) was able to recover anaerobes from approximately 35% of the specimens received in their laboratory. The ratio of anaerobes isolated per specimen was 1.4 to 1. A summary of their results is given in Table VI. The incidence of various anaerobes as normal flora in humans is given in Table VII (Sutter, 1972). No mention is made as to the exact procedures used to gather this data. The ~naerobes from human infections most frequently submitted to the Center for Disease Control (CDC) anaerobe laboratory are given in Table VIII (Dowell, 1972). The most common species of anaerobes recovered from clinical specimens reported by naore 0'·1001"'0, personal communication) are: No. % B. fragilis 30 22 C. perfringens 15 11 PSt magnus 10 7 E. lentwn 8 5 Ps. intermedius 5 4 0. cor\"odens 3 2 E. 1 imosum 3 2 P. acnes 3 2 He suggests that one concentrate on knowing these organisms because they comprise approximately 50% of all the anaerobes isolated from clinical specimens. TABLE VI Summary of Anaerobic Bacteria Isolated According to Strain and Source 1971 ) c;; .5 Bacteroides fragilis 17 70 92 1 25 233 91 25 212 122 121 0 14 17 1,051 21 B. melaninogenicus 13 25 3 12 37 29 13 50 24 0 0 0 1 55 289 6 Bacteroides sp. 7 44 58 2 12 97 57 21 87 59 15 5 2 7 36 410 10 Fusobacterium fusiforme 13 10 23 I 6 35 17 13 37 21 7 1 2 0 39 4 Fusobacteriunl sp. 2 I 1 8 0 8 30 10 4 34 16 2 0 1 0 14 145 3 sp. 14 18 6 0 4 24 5 6 30 9 ""/.. 1 1 18 139 3 Peptococcus sp. 70 212 37 2 12 76 69 38 179 95 3 8 5 23 J3 842 17 Peptostreptococcus sp. 15 49 29 2 12 73 50 25 81 50 9 2 3 5 22 427 8 Clostridium perfringens 17 20 0 10 50 10 7 34 10 2 0 2 6 249 5 Clostridium sp. 13 8 11 1 8 3 5 20 17 22 2 0 2 136 3 Propionibacterium acnes 42 117 4 2' 89 29 40 40 129 155 146 6 9 6 14 828 16 Propionibacterium sp. 3 5 3 0 8 11 3 0 6 10 2 0 0 0 1 52 1 Eubacterium sp. 1 5 3 0 7 6 0 6 5 0 0 0 1 36 1 N ..s::=. TABLE vn Incidence of Various Anaerobes as Normal Flora in Humans (Sutter. 1972) Clostri- dium Non-sporulating bacilli Cocci Gram-positive Gram-negatIVe Propion· Actino- Bifidobac- Eubac- L'lcto- ibac- Bacter- Fusobac- Gram Gram myces tcrhur. tcrium baciHust tcrium oides terium Vibrio positive negative Skin 0 a 0 u 0 2 0 0 0 1 0 Upper respiratory tract* 0 1 0 T. 0 1 I 1 1 I I ") Mouth + 1 1 1 1 + 4- 2 1 2 2 Intestine .2 + 2 ""L 1 ± 2 1 + 2 I External genitalia 0 0 0 U 0 U 1 I 0 1 0 Urethra + 0 0 U ± 0 I 1 + ± u Vagina + 0 2 U 2 0 I + I 1 1 * :::::includes nasal passages, nasopharynx, oropharynx and tonsils U= unknown o :::::not found or rare ± :::::irregular I = usually present 2 :::::usually present in large numbers t = includes anaerobic, microaerophilic and facultative strains f\:) 01 26 TABLE VIII Anaerobic Bacteria from Iluman Infections Nost Ft~el1uently Submitted to the CDC Anaerobe Laboratory (Dov:e 11, 1972) 1. Clostridia C. bifermentans C. butyri cum C. cadaveris (C. capitovale) C. innocuum C. limosufi1 (Clostridium sp CDC group P-l) C. perfri ngens C. ramosum (Catenabacterium filamentosum Bacteroides terebrans) Cw septicum C. sordellii C. sporogenes C. subtc~rm;nale C. tertium 2. Non-sporeforming grar.1-positive bacilli ActinOMYCeS isr~elii Acti nOllyces odonto lyti cus ActinoQYces naeslundii J\rachni a propi orri ca (/\ct -j nomyces prop; OIri ells) nifidobacter"j U8 eri k~oni i (Actinomyces er; ksoni i) Euhacte riuM a1 acto lyt; CUrl (Ram; b acter'; um s peci es ) Eubacteriul1 lenturl (Corynebacterium diphtheroides) EubacteriuM liDosum Propionibacterium acnes (Corynebacterium acnes) Propionibacterium granulosum (Corynebacterium granulosum) 3. Non-5poreforming gram-negative bacilli Bacteroides fragili~ S5 fragilis (B. fragilis) Bact0roides fragilis 5S thetaiotaomicron (8. variabilis) Bacteroides fra9ilis 5S vulqatlls (B. incommunis) Bacteroides melaningogenicus ss asaccharolyticus Bacteroides mclan;noqenicus 55 intcrnedius Fusobacterium mortif~ru~ (Sphaerophorus ridiculosis) Fusobacteri Wil nccr'ophorurn (Sphact"ophorus necrophoru5) .Fusobacterium nucleatum (Fusobacterium fusiforme) 4. Anaerobic cocci Peptococcus sp CDC group 2 Peptostreptococcus sp CDC group 1 PeptostreptococCU5 5p CDC gr'oup 2 Peptostreptococcus sp CDC group 3 Veil1onel1a alcalescens - Veil10nelln parvula III MATERIALS AND METHODS A. Sources of specimens. All of the clinical specimens used in this paper came from patients at the University of Utah t1edical Center. Because of the limited capacity of the laboratory and the lack of experience in dealing \'Jith anaerobes, it was decided to select the specimens that would be accepted for culture of anaerobes. The problem of collectii19 the speci mens ViaS also a reason for the contro lover the types of s peei mens that \vould be: accepted. He did not \'lant to get into the problems of spending a lot of extr'a time and media isolatinq and identifying organisms from specimens contaminated \'lith normal flora. Only specimens from abscesses., putrid infections, body cavities \vhich appeared infected, pelvic, wound infection and any suppurative infection that did not respond to treatment were accepted. Those specimens that vJet'e not considered acceptable "Jere sputum, br'onchial washings, urine, except those, collected by supra pubic bladder aspiration, stools, vaginals and wounds freshly or continuso~ly contaminated \\fith normal f'lor'a. These spec'imens v!ere excluded because they contain large number's of anaer'obes as nOl"mal flora. It \'1ould be beyond the capabilities of our laboratory to make a study of the normal flora. If there \Vas any other type of specimen that the physician felt warranted anaerobic culturing, it was referred to Dr. Jam(;$ N. ~Jilfert of the Infectious Diseases Division for approval. 28 Several organisms \'Icre obtained from Dr. S. r1. Finegold, Hadswortll V. A. Hospital. Los Angeles, California. Several other organisms were received from the Center for Disease Control as unknowns. B. Tra,nSRort Methods .• The collection procedures employed in any anaerobic bacteriology laboratory are among the most important parts of the total system if one is to recover significant numbers of anaerobes. If the organisms are lost before they get to the laboratory there is no system that is good enough to resurrect them. A vigorous attempt was made to collect all specimens in the best manner possible and to have them cultured as soon as possible after theil" arl~ival in the .laboratory. Ana-Port vials and Ana-Swabs (Robbin Lnboratories) were the two principle methods used for collection of specimens. These h/o systems \'Jere made availahle in the clinical labor'atol"Y to allY physic-ian that v/anted to collect a speci men fot" anaerobic culturec They were not made generally availa.ble in the vIa rds and in the operating room because it \las felt that more can tro 1 \'Jas needed over their use since they required some special instructions. Hhcn someone came to the 1aboratory for the co1lection l:lut.er·ials they viera given brief explanation as to hO\,I they vlere to be used. In this \IJay. He VICH'e able to communicate vJith the doctors that were using this service the importance of proper collection. Another important reason for the control of the collection systems was that they had a shorter shelf life than the other methods and there ,',as no contr'ol over hm'J long they remained on the shelf ; n other areas of the hasp; ta 1. Th; S \'/ay \'Ie \A/cre assured of fresh 29 collection media. The resazurin indicator \1ould change in a relatively short time and \'/ould have to be replaced \'lith a fresh tube or vial. Ana-Ports arc designed for collecting specimens that can be aspirated. They are small serum vials containing carbon dioxide and resazurin as an indicator of the presence of oxygen. A small amount of water is added to effect sterilization in the autoclave. The specimens were collected in a syringe and injected into the vial. being careful not to admit any air. If done properly, the indicator did not change co lor. Thi s vIas then sent immedi ate 1y to the 1aboratory for plating out under anaerobic conditions. If this system \'/aS used proper ly, the specimen was never exposed to air and no loss of clinically significant organisms resulted. For specimens that could not be aspirated, such as pieces of tissue, bone~ or small amount5 of drainage that could not be collected in any VIaY other than a sV/ab or ina contai ner the Ana-Svtab vIas used. This cons i sts of tHO 16 x 150 mm tubes fi tted\AJi th a No. 1 butyl rubber stopper. In one of the tubes is placed a cotton swab. Each of the tubes has been gassed Hith oxygen free carbon dioxide and sterilized under anaerobic conditions. Resazurin is added as an i nd; cator" of the presence of oxygen. As Hi til the Ana-Ports ~ a sma 11 amount of \later has been added to effect proper sterilization. Hhen the specimen was ready to be taken. the cotton swab was removed from the first tube and used to collect the specimen. The swab was then rapidly inserted into the second tube. keeping it open only long enough to insert the swab and avoiding prolonged exposure to air. Hheo done properly, this method \'Jas adequate for the commonly found 30 anaerobes in clinical specimens 4 More care must be exercised with this system than the other and was the method of second choice. Occasionally a specimen \'/aS sent to the laboratory that had been collected by the routine procedure using Stuart's transport media. This vias discouraged for several reasons. The amount of transport medi a that is rout; ne 1y used in our 1aboratori es \A/as such that sma 11 amounts of anaerobes would be diluted out and made difficult to recover. Facu1 tat; ve organ; srr'Js v/oul d have the opportuni ty to overgro\,/ the anaerobes if not cultured immediately. The media is made up several days or weeks prior to use and as such would not be reduced enough to a11 o\'/ surv; va 1 of the more oxygen sens; ti ve anaer'obes. Us; n9 routine collection procedures for anaerobes would tend to let personnel handle them the same as the routine bacteriology work and therefore \'1ould not be plated out irnrnediately as recommended. Specimens that had been collected in syringes and capped and sent to the laboratory as soon as possible ",ere accepted for culture. Those collecting the specimens were cautioned to exclude as much air as poss i b1 e from the syri nge before cappi ng it and vJere encouraged not to aspirate any air. All specimens VJel"'e sent to the laboratory as soon as possible.. Care \vas taken to instruct all laboratory personnel that specimens should not be incubated or refrigerated befor"c being plated out. Incubation allo"/s the overgrm'/th of the facul tati ve organ; sms and therefore \·d 11 make it more di ffi cu1 t to recover any anaerobes pl''Iesent. The 1o\'ler ter.1peraturc allO\vs more oxygen to dissolve into the media and therefore ltlill change the flora of the anaerobes. 31 c. 1.501 at; on I7lC thods. There were two anaerobic systems commercially available that vlou1d fulfill the need of this study, the anaerobic glove box and the roll tube apparatus. Both have been shov/n to prev; de adequate anaerobi c conditions for extremely oxygen sensitive anaerobes. Both have some advantages and disadvantages. 1. Anaerobic glove box. An anaerobic glove box \'las obtained from Coy f.1anufacturing Co., Ann Arbor. Michigan. It was designed bi Dr. Rolf Freter (Aranki. 1969). It consists of the following: An anaerobic chamher, 84 x 32 x 40 inches made of 20 mil pressed polished clear vinyl with a 40 mil frosted vinyl bottom extending two inches up on all four sides. One nipple. one and one ha 1finches 0.0., fi tted \'Ii th a rubber stopper \,/11; ch had holes in it to permit the passage of electrical \'lires inside the chamber e Since our laboratory trains f.lany people each year, including medical technology students, \'/e had placed in our glove box two pairs of gloves instead of the standard one pair. The gloves were size nine with GL 20 sleeves. The sleeves were permanently attached to the chamber; the gloves were replaceable in case of accidental puncture. The v/ho1 e chamber \las r;'-ounted on a 3/4 inch ply\-1ood base that had been covered vii th a 1/4 ; nch foam pad and a heavy vi nyl coveri ng. A one inch tubular aluminum rack suspended the chamber. The sides of the chambe~ were slanted inward slightly for convenience in working. If they v/ere straight up and down the \!/orker \vou1d have difficulty in keeping his nose out of his work. The entry lock \'las made of clear anodized a1uminum. It \'las fitted wi th t\,/o doors, one on the ins i de and the other on the outs i de of the 32 cha~ber. Each door had a locking mechanis~ and a soft neoprene a ring to seal the door. The inside 0 ring was glued in place using Silastic 732 RTV Adhesive Sealant. The outside 0 ring vias not glued in place because it is necessary to have a \iJay for gas to escape in case the lock or chamber is accidentally pressurized. To preserve anaerobic conditions in the chamber, the gas must not enter it from the airlock unless it has been properly flushed. The reason the inside 0 ring was glued was because it is difficult to replace because the gloves are cumbersome to \-Jork vJith. The airlock \'Ias assembled \vith a pressure vacuum guage and two high vacuum ball valves. There Vlere t\,/o catalyst boxes in the glove box. Each had a metal tray to hold the catalyst. The bottom of the tray ViaS made of stain less steel wire mesh to allow air to circulate around the catalyst. Each box \'/dS fi tted vJi th a "muffi n fan ll to ci rcul ate a; r over' the catalyst. One of the boxes had a heater that \'/aS in continuous operation. It Has not enough to maintain the temperature of the glove box at i ncubati on terJ1perature so the second box contui ned a thermostatically controlled heater' that regulated the temper'ature. Because of the continuous introduction of moisture in the form of media and reagents and the reaction of the hydrogen and oxygen, a dessicant vIas required to remove it from the atmosphere., The dessicant used VIas Tel Tale Silica Gel (Davison Commercial Chemical, Baltimol"e, r-laryland). This dessicant vIas rejuvenated every other day. This was done by heating in a drying oven at 80°C overnight. This dessicant had an indicator in it that changed from a blue color to a bluish pink to pink when exposed to moisture. 33 The gas mi xture recommended for use in the chamber vias a singl e mixture of 10% hydrogen, 10% carbon dioxide and 80% nitrogen. This mixture is not available in Salt Lake City. The cost of shipping it from the nearest supplier was prohibitive and the problem of getting the gas when needed was too great. We elected to use a mixture of 10% hydrogen ;n 90% nitrogen (Hhitmore Oxygen Supply, Salt Lake City) as the source of hydrogen to react wi th the oxygen. t'Je used a second tank "lith only carbon dioxide (Hhitmore Oxygen Supply, Salt Lake City) for the source of carhon di oxi de for grov/th requi rements and stimulation. The catalyst used was obtained from Engelhard Industries, Vineland. NevI Jersey() It \lIas Deoxo Palladium coated alumina catalyst type D. We used 1/2 pound of catalyst for each of the boxes. The catalyst was rejuvenated, in the same manner as the dessicant. evcl"Y b'JO days. To assure us of anaerobic conditions. an anaerobic indicator strip (BBl) was used. Also. resazurin was added to all media where possible to check for exposure to oxygen. In order to uflame" loops and flasks inside the chamber an incandescent fl ami ng devi ce \,/as used (Bact i c inerator. Shervlood f·1edi ca 1 Ind •• Inc.). The vacuum pump used \'Jas a Duo-Seal model 1405 (He1ch Scientific CO. c Skokee, Illinois). For a complete picture of where things were placed inside the glove box see Photograph 1~ To introduce a specinen or other material into the glove box. it was placed inside the airlock and the doors sealed. The vacuum pump Photograph! - AnaeJobic Glove Box Used at the University of Utah w .,J:::oo 35 that was used did not create enough vacuum. so we had to evacuate and flush the airlock three times. After the first two evacuations, the airlock was filled with the carbon dioxide. After the third evacua tion. the airlock was filled with the hydrogen and nitrogen mixture. This procedure pt"ovided adequate hydrog(:n for reaction '(lith any oxygen present and supplied sufficient carbon dioxide for the growth of ol"gani sms. For tubes with stoppers, clamps had to be used to prevent them from popping off while under vacuum (Bel·leo Glass Co., Vineland, New \Jersey) • At one point. the glove box became contaminated with large amounts of oxygen. Attenpts Viere rlade to introduce sufficient hydrogen to react with the oxygen but this proved useless and it became necessary to dismantle the entir'e frarae and allo\'J the plastic box to collapse and push out as 1.1uch air as possible. At this pO'int. the chamber was inflated with the mixture containing hydrogen and allowed to equilibrate for two days, 2. Roll tube appara,tus. The roll tube apparatus was purchased froM Belleo Glass Co •• Vineland. ilevi llersey. The ind"ividual components to it are as follm'/s: 1. Motor for rotating tubes to be streaked. 2. Clar;lp for holdinn the tubes while being streaked. 3 •. Gas cannul as for steri 1e. oxygen free car'bon di oxi de to enter the tubes while they are open~ 4. Y tube to separate the stream of gas into the cannulas. It also contains a cotton plug which has been sterilized to filter the gas as it passes int0 the cannulas. 36 5. Oxygen free carbon dinx'ide (National Cylinder Gas, Salt Lake City~ Utah). 6. Foot peda 1s \'/h i eh a re attached to items 3 and 7. 7. A syringe used to inoculate biochemical tubes rapidly. 8. The Pasteur pipette is connected to the syringe for delivery of the inoculum. See Photograph II for the roll tube aparatus. Tubes that were to be streaked for isolation of colonies were placed under the cannula that had the motor under it. Care was taken to be sure that no air entered the tubes when placing them under the stream of gas. This \'Jas accomplished by holding the tube close to the cannula \"/hile removina the stopper. The stoppers vJere removed by using a modified hernostat. A six inch hemostat vIas bent to fit the butyl rubber stoppers, This ViaS accomplished by heating and bending vJith a pair of pliers. This allowed us to remove the stoppers that have been flamed and are warm without any discomfort and with a minimum of contamination. Bi ochemi ca 1 tubes ",ere inocul ated by l:leans of the syri nge and Pasteur pipette. The Pasteur pipette \'Ja5 filled by placing the tip into the cultur'e broth and aspirating \,Jith the syringe. The pipette vIas then pl aced above a cannul a. The tubes to be inocul ated \'/ere placed under the pipette and the foot pedal was depressed turning the arm attached to the syringe pushing in the barrel and alloHing three or four drops of the ·inoculum to drop into the tube belov/" t1any tubes could be inoculated in a short period of time in this Qanner. 37 38 D. I den;;'; fi cati.Q!l of anaerot~e2... 1. Gas chr~atography. The gas chromatograph used vIas a dual column, i sothenYla 1 thermal conductivity (Dohrmann Envirotech, f:1ountain View, California), fitted with a six foot by 1/2 inch aluminum column. The column packing used was 20% (wt.) Resoflex (LAC-I-R-296, Burrell Corporation, Pittsburgh, Pennsylvania) on 30/60 mesh Chromosorb. The operating conditions were as follo':/s: l. Attenuator: lX 2. Detector current: 95 milliamps 3. Column temperature: 127°C 4. Injection port temperature: 150°C 5.. ~lc 1 i um carr; er gas fl m'l r"a te: 120 cc per m; nute. The rccor-der vIas a Doh r'mann node 1 flu·300 poten t i omet ri c s tri p cha rt recorder. single channel, horizontal chart platen, one millivolt span. The churt paper "las eleven inch calibrated \'lith r.lajor time lines every inch (Dohrmann r!04/ 704204). Hhen run at the s 10\'/ speed setti nf] tone inch equals tvJO m"inutes. All recordin9s vJere done at the slol;'/ speed. Because of the long peri ods of time necessar'y to equi 1 i brate thi s type of gus chrOl"fatograph t the equi pmcnt vIas 1eft on a 11 the tir:1e, \,Ii th the exception of the detector current and the recorder~ When not in use, thG flo\,1 rate of the carl"'; EH' 90S ViaS reduced to on ly a few cc' s per minutejl to reduce the amount of heliUM consUlned., This provided enough flmv to prevent oxygen from diffusing into the detectors and burn; nQ out v/hen the current "laS turned on. About ten mi nutes before 39 use. the detectors \Jere turned on and the gas flo\l/ rate adjusted to 120 cc per mi nute and all olf/ed to equi 1i brate. The colur:ns v/ere packed in the fol10vling r:1anner. After the aluminum tubing was shaped into the desired configuration, one end \'las plugged \'/ith a small \'Iad of glass \'/001 (Pyrex flo. 7220) and this end was attached to a vacuum source 0 ~'Ji th the column 1y; ng f1 at on a counter. the packing materi a1 \,/as poured ; nto the open end "Ji th the aid of a small funnel. As the material was added, the column vas sharply tapped ~";th the handle of a scre\lldr;ver to help pack the material evenly into the tubing. Caution had to be exercised to prevent too vigorous pounding with the screwdriver and packing the col umns too ti ght. J\fter the tub; n9 vias fi 11 cd \I the other end vIas plugged with a second wad of glass wool. When a new colurm was put in or the line opened in any way. the syster.l had to be checked for 1eclks of the carrier Qas. Thi s was done by removing the exit ports and replacinq them vlith a special plug prey; ded by the manufactu}"el". The plug all m'/ed press ure to be bui 1t up in the system. The regulator\'Ias then brought up to about 50 pounds and left for a minute. After the regulator was turned off, the pressure should have been stable. A drop in pressure indicated a leak in the system and it vIas detected by brushing on a solution of 1i qui d soap and \!ater and observi ng for hubb 1es" The chromatograph was standardized using standard sets of acids and alcohols (Dohrmann Envirotcc. f,1ountain View. California)" The standard acids contained the follmving acids in the given concentra tions in \veight percentages: 40 Acetic 5.5 Propionic 5.2 Isobutyric 5,.0 Butyri c 7.6 Isovaleric 9.7 Valerie 19.7 Isocaproie 8.4 Capl~oi c 19.5 Heptanoic 19.4 The standard alcohols contained the following alcohols in the given concentrations in percent by weight: Ethanol 9.3 Propanol 14.2 Isobutanol 14.2 Butano1 14.3 Isopentanol 14.3 Pentano1 14.4 Hexanol 19.4 Extraction Procedures The procedures folla1cd to prepare a sample for analysis of the various volatile fatty acids are as fo1loV/s: 1. 2 ml of the culture was acidified with three drops of 50% aque- ous H2S04- 2. Add 1 ml of ether. Stopper and invert 20 times gently.' 3. Centrifuge to break the ether-water mixture. 4," Place in a freezer until the water layer is frozen solid. Pour the ether layer off into a small tube (12 x 75 tml) and add 41 anhydrous f'1gS04 to equal about one half the volume of ether and stopper and let stand at least ten minutes in the freezer or refri gerator. The HgS04 removes any res i dua 1 \-/ater in the ether. The reason for acidifying the fatty acids is that the salt forM is soluble ;n water but not in ether. At 10\,1 pH the acids are in the free acid form which is soluble in both water and ether. The reason that after adding the MgS04 the sample was placed in the freezer or refrigerator for ten minutes instead of room temperature. was that if the sample ViaS aspirated into a syringe \'Jhi1e wan'l the 'ether would vaporize and all you would get in the syringe would be the ether vapor and very littie if any ether extract. The syringe was placed in a cool place for the saMe reason. t-':ethylation of the nonvolatile fatty acids such as lactic and succ-inic acids f Has performed in the follov/ing manner: 1. One ml of the culture was added to a disposable glass, 12 x 75 tube and acidified ~ith one or two drops of a 50% H2S04 solution. 2. Two ml of methanol added and 0.4 ml of the acid solution added. 3. Stopper and heat in a 55~C water bath for 30 ninutes. 4. 0.5 ml of chlorofol"ln added and nixed by inversion 20 times. Centrifuge for a brief period to break the emulsion. If the emulsion does not break, one cc of water is added and mixed by inversion and the process repeated. The chloroform layer is the small layer on the bottom. 42 Analysis for the above mentioned of the Methylated products was requi red for the gram negati ve rods that di d not shO\'1 a maj or butyri c acid peak. It was also required for the gram positive rods and cocci if: 1. No aci ds \1ere detected Hi th the ether extract 2. Only acetic acid was detected with the ether extract 3. Or if only acetic and formic acids were detected with the ether extract. Samples were introduced into the gas' chromatograph by a 50 micro~ liter capacity sample introduction syringe \'lith a Teflon type plunger fi tted 'iIi th a one inch long. 24 gauge. 0.012 inch i. d. cemen ted needle with 17° nancoring tip, and Chaney. adaptor for reproducible sample. introduction (Hamilton No. 1705-SNCH). The sample size was 14 microliter. The volatile fatty acids \'Jere introduced 'into the upper" column and the methy'Jated nonvolatile fatty acids {'1ere introduced into the 10\1er column. 2" lledi a usp..d" There were bvo types of media used: media for isolation and media for identification. The isolation Media are divided into tHO additional groups. one for the glove box and one for the roll tubes. The media used in the glove box were preparea in the regular manner arid placed in the glove box for 24 hours to reduce it before use. a • r~e di a for I sol at; 0 n The nonselective medium used in the glove box was sheep blood agar made from Columbia agar base supplemented with 0.2 ml per liter Hykinone (Abbott). a co~mercial preparation 43 of vitamin K. The blood was laked by repeated freezing and thavling. Laking of the blood allo\'Js more rapid detection of the typical black colonies of !!... melaningoqenicus. Selective media included Paromomycin (100 mcg/ml) Vanco myc'in (7.5 mcg/ml) laked blood agar (PVLB). This vIas used as a selective medium for isolation of anaerobic gram negative rodsi the Vancomycin inhibits most of the aerobes and the Paromomycin ;nhibitsmost of the gran positives. This medium was selected because of the high incidence of facultative gra~ negative rods and the difficulty in separating them in culture, Phenylethanol agar was also used as a selective medium. Thi s medi urn all ov/cd the grovJth of a11 the anaerobes and the aerobic gram positive cocci, The only group of organisms that is is inhibitory for' is the facultative gram ne~Jative rods, Using these three medial! it "las felt that \'/e \'lOuld be able to recover most of the common anaerobic organisms that would be encountered in the spec; mens that \Ie \'/ere to recei ve. The PVLB medium has been recoror:'lended by Finegold as the selective medium of choice for the anaerobes when only one type of selective ~edium can be used (Dlair i Lennette, Truant, 1970). The basic medium used in the roll tubes was supplemented Brain Heart lnfus'ion agar (m-tIS). From this basic medium the selective Media were made by adding the appropriate materials, The BlUS \-JaS prepared in the folloVJing manner: 1. Commercially prepared dehydrated brain heart infusion agar was rehydrated according to the manufacturer's directions. 44 2. Four M1 per liter of resazurin stock solution added. 3. The mixture was boiled until the reSazurin indicator turned colorless. 4. One ml of the hemin solution was added per 100 m1 of med-j urn. S. 0.2 ml of Hykinone (Abbott) added per liter. 6. The mi xture vias placed under a stream of oxygen free carbon dioxide. 7. The medium was dispensed in 12 ml amounts into gassed out 25 x 150 mr:l roll tubes and capped \,/i th a Ho. 1 butyl rubber stopper". 8. The tubes were than placed in a press (BelleD Glass Co •• Vineland, N.J,) and autoc1aved at 121° for 20 minutes, 9. After removing fron the autoclave and cooling to 55°. the tubes were spun horizontally rin the roll tube spinner (Bel lee Glass Co.) until solid. 10. All anaerobic media were stored at room ternper~ture until used. The resazurin stock solution \'Jas made up by dissolving one resazut'in tablet (ca. 11 mg, Allied Chemica', Catalog No. 506) in 44 ml of distilled water~ The stock hemin solution was made by dissolving 50 mg hemin in one ml of 1 N sodi urn hydrox; de and 'di 1uted \-J; th dis till ed water to 100 ml. It was then autoclaved at 121 0 for 15 minutes. To make the selective media, appropriate ingredients listed belovl v/ere added to the autoclaved basal medium before 45 spinning. All procedures \rlere carried out under a stream of oxygen free carbon dioxide. Phenyl ethyl alcohol anar. 0.03 m1 of phenyl ethanol \Alas added to the 12 ml of basal medium in the roll tube and spun on the roller until hardened. PVLB agar. This \'las made by adding 3-4 drops of laked blood to the melted and cooled medium in the roll tube and then 0.25 ml of a sol~tion containing 0.48 grams per 100 ml Parornomyci nand 0.036 grams per 100 rnl Vancomyc; n to make , a final concentration of 100 r.1cg per ml Paromomycin and 7.5 mcg per ml of Vancomycin. b. Media for Identification' All of the tubed media used for identification of the organisms was purchased fro~ Robbin Laboratories, Chapel Hill. North Carolina. The media included the following prereduced anaerobically sterilized media. Chopped meat ca,,'bohydrate Peptone Yeast Extract Glucose (PYG) Peptone Yeast Extract (PY) Al"abi nose Bile Esculin Fructose Lactose f1annose r~ann; tol Raffinose 46 Nitrate Gelatin Chopped meat agar slants BHIS agar deeps Rhamnose Trehalose Starch Xylose tvta 1tose Sucrose Lactate Threonine Occasionally other media were required for identification and these Here made up (\5 needed. The mas t cornman ly needed ViaS egg yolk agar for identification of Clostridia. Prereduced BHIS in 16 x 150 mm tubes was clamped \·lith a special clamp (BelleD Glass Co.) designed to keep the rubber stoppers in place while heating them.· Hhile the agar \'Jas being melted. the petri dish was opened and 0.5 cc of egg yolk suspension (COLAB) was placed in it. After the agar was cooled, it was poured into the plate and swirled to get an even suspension of the egg yolk and allm 3, ~dentificutio~ procedures. The procedw"es for identification of the anaerobes fol10\,/ed closely that of the Virginia Polytechnic Institute (Holdeman. 1972). Because the media \~as so expensive ($0.47 per tube) several modifications 47 had to be made in the schenes recommended. After the organisms had been isolated, a gas chromatograph pattern Has run and on the basis of that pattern the appropriate biochemicals were then selected. Instead of inoculating all of the recommended media, we used only the media necessary to identify the most common of the isolates in each group_ In ttli s manner. we were ab 1e to cut dovln on the number of tubes used. If an organism was isolated that was not the organism expected II then a de 1ay in; dent i fi cat; on vIas encountered because more media had to be set up and incubated. The bas; c sal ecti on of medi a is as fo 11 OHS for' each group of organisms. Fusobacterium,...... Propionib~cterium Esculin Nitrate Lactose PYG fhreonine Gram Positive Rods PYG PYG Bile f4anni to 1 Bacteroides Fructose PYG f1a 1tose Fructose Chopped meat agar slant Bile Clostridium Trehalose Egg yolk agar plate Rhamnose Gelatin Gram Positive Cocci PYG Lactose Lactose r1a 1tose Sucrose Fructose Chopped meat agar slant 48 Gram Negative Cocci Nitrate PYG The procedure fol1ol,rJed for· handling the specimens varied depending on the flora recovered and the source of the specimen, but in general the follO\ving schedule \",a5 fo1loy/ed: Day 1 Streak the specimen on roll tubes containing the three media--BHIS, PEA and PVLB. Day 2 Pi ck each colon i a 1 type to chopped meat ca rbohydrate in the morning. A1lov/ these to incubate the, remainder of the day and then subculture each to an aerobic and an anaerobic blood agar plate to determine the oxygen tolerance of each isolate. Day 3 Any anaerobes are gram stained and a gas chromatograph pattern obtained of the volatile fatty acids. On the basis of this information, the proper selection of biochemicals was chosen and inoculated. Day 4 Read the biochemical reactions and determine the identification. If necessary t a methyl ated extract \',/as gas chromatographed. If any additional information Has necessary for identification, the proper tubes were then inoculated. All data Here recorded on forms developed by the Virginia Poly technic Institute (Holdeman. 1972) (See Page 51). All the reactions v/ere recorded on the ri gh t hand margi n of the chart and cou 1d be 49 laid over the reactions for the particular organisms for comparisons. The indole test \'Ias performed on all isolates by adding approx imately one cc of chloroform to one of the carbohydrate tubes, usually the PYG. and shaking the tube. One dropper full of Kovac's reagent \'/a5 then added and mi xed '-"Ii th the cll 1oroform 1ayer. A positive test shml/ed a bright red color developing in the chloroform layer in the tube. The motility test \'las performed by removing a portion of the actively grovdng culture \'1ith a Pasteur pipette and placing this on a microscope slidee The slide was immediately covered with a cover slip and placed under the microscope and observed for motility. Since atmospheric oxygen will inhibit motility of anaerobes, the procedure had to be carried out as rapidly as possib1ee The nitrate test was performed by placing one dropper full (appr-oximate1y 1 m1) of reagent \lA" in the tube and then adding one dropper full of reagent "[3" (Baily, Scott, 1971). A positive test was read as a dark red color developing .in the tube e Sometimes a red precipitate was observed and this was interpreted as positive also. All of the fermentation reactions "Jere read using a pH meter. We used a Sargent Welch pH meter (Model LS, Sargent Welch Catalog No. 5-30005) fitted with a single combination e1ectrode(Sargent Welch Catalog ~!o. S~30072-15). The electrode was thin enough to fit into the tubes to be read and moved frum tube to tube rapidly. The results were interpreted as fol1m'/s: pH 6eO and higher \'las regarded as negative. pll 5.5 to 6.0 \'/as considered as \'leak. Clny reading 5.4 50 or less \'Ias interpreted as pos i ti ve. On arabi nose, ri bose and xylose the pH of a negative organism read lower than the other sugars and the negative reaction Vias read from pH 5.8 and higher. 51 ______Ident ______-rpt. Datc-reed. gr. pH/rc(lction ~3nle ______-- __-- ______* PY • Source ------amy~:u;:din Diagnosis ______• arabinose ." cellobiose + Stain: Orig. Single col. erythritol .. esculin pH + _T5"I esculin hyd. -- --~P='" * fructose Streaks: glucose Anaero. * + glycogen inositol '* betosc :~ mJltos~ Aero.1"S ~==""='----- ~--=~=",,,,"' :! mJnnitol , •_s man no:;;:! .. (°1 Broth gf. .. + s Grov"t:l in:, PYG. ______ pyC; Twecn+· ____ 0.1°2~ Rt::duc. _.____ .. _____ "0 rh~l 5 · PYG--Bile·~ ______.___ 5, G:1S+- - ribose <"J PYG-lkme (K)- Color salicin Pyruvate Urease (51 ______+ sari" Lacl::ltc-· T Ilea t rcsist. t _5 starch TIuconine - (0' ___ • P ____ Fbgella ______star. hyd. sucrose T Glycine trehalose GCA Prod.: PYG* ______. ______~y _sof - gel3.tin milk •s m~at * indo! f--- .. nitr:He +- catala~H' bik g,r. of 1-, S leci th inase hemolv:-:i" ------.-f--.--'~- ~-. U-..e f(H: .~ all: + '" ,ram-p\H,tivc NSF fOch; _ '" f.lam·r.er.:ltJvc NSF rOd1;"" ,oeci; s" sporing motility ,(xII; T C (tc.;>(WCI1Ie!. I 1= Ilsullly not l~qU\l~J. FOlr': USED FO R RECORDI NG· HESUL TS IV RESULTS A. Results of qas chromatoqraohy. The elution times in minutes for the standard volatile fatty acids on the gas chromatograph are as foll O\,/S: Acetic acid 5.8 Formic acid 6.7 Propionic acid 8.5 Isobutyric acid 9.5 nutyric acid 1207 Isovaleric acid 15.2 Valerie acid 21.0 Isocaproic acid 28.6 Caproic acid 34.3 Heptanoic acid 55.4 Capryllic acid 75.0 Hith the methylated det"ivatives. the only tv/o products that are of value in identifying anaerobes seen in clinical specimens are the lactic and succinic acids. The elution times of the methyl esters of these acids are 2.4 minutes for the lactic acid and 13.1 minutes for the succinic acid. 53 In the graphs of the gas chromatograph patterns, the first one (A) is the volatile fatty acids and the second one (8) is the methyl esters. Graph number one shows a typical !Jas chromatograph pattern for !til ,frani 1i s 5S fragi 1is. The aci ds detected \-/ere aceti c, propionic, and isovaleric for the ether extracted acids and large succinic acid peak for the methylated derivatives. The major peak of succinic acid is very characteristic of this organism. Graph number t\-JO is another !!.. fraq~ ss fraQi 1is. The aceti c ac; d peak is larger than the propionic acid peak with this strain. but the major peak of succinic acid is the samac Graph number three shaHs the patterns for a typical Q.. melanino geniClJ.5 S5 2.§sachar.olyticus and has acet'ic, propionic, isobutyric, and isovaleric acids. No methylated derivatives were made with this organism since they at'e not necessary for identification& Graphs four and five are of organisms of the genus Fusobacterium. The large butyric acid peak separates this genus from the genus Bacteroides. They also do not have the large succinic acid peak on the methylated derivatives. Graph six is that of Veillonella alcalescens. Both species of Veillonella have simila," gas chromatograph patter-ns. Occasiona11y during the study, a gran positive cocci \'/as over decolorized during the gram stain and called a gram negative cocci until the gas Cht~Ohlatograph ViaS run. None of the common gram pos i ti ve cocci wi 11 give a pattern like this. The nonspore forming gram positive rods gave patterns like those shovin in graphs seven to eleven. The 1arge 1act; c aci d peak is 54 55 UIlOJOlOll1J 56 57 lliJOJ010l1D 58 59 [ 60 UllOJ010 llO 61 c > I: 62 63 64 .------ UllOJOJOIlfJ 65 characteristic of the lactobacilli (Graphs 7-8) and the major peak of propionic acid is characteristic of the genus Propionibac terium (Graph 9). Organisms froM the genus Clostridium gave a variety of patterns. £. perfrinqens produces several acids and gives a pattern such as in Graph 12. The methylated extracts are not helpful in identification of the Clost~idia and were not done routinely. The gram positive cocci produced patterns such as the ones in Graphs 13-16, the most interesting being the pattern for ~ryto streptococcus pnaerobius because of the very typica1 pattern. Hith the gram morphology and this pattern for the gas chromatograph no other tests were needed for identification. B. .9!Lt.ure rc~u 1ts, Table IX sho~s the total number of specimens studies by source and the number of anaerobes and aerobes recovered from each. Eighteen percent of the specimens submitted had no gro\,/th. Of the cultures that had grm/th. 92.5% had anaerobes present, either in pure or mixed cu1ture. 64f!S;S had aerobes, 46% had mixed aerobic and anaerobic flora. Of the anaerobes orqanisms, 65.9% were not mixed with other anaerobes, 18.2% v/ere found \1i th tvlO anaerobes, 4.5% had thl"ce anaer'obes and 11.4% had four or more anaerobes in the same specimen. Table X shm1s the number of organisms from each source cultured and the percent of the \vhole. either aerobe or anaerobe. Also given is the number of each type of specimena dn its percentage. I .. coli vias the mos t common aerobe and .!!.. fraq;' i S \'/as the mos t common anaerobe •. The mas t common source of each of these t\'10 organ; srns \-/as from abdominal specimens. 66 [ 67 68 ~ .~ ...... o E cy I-. ....l J?, 2 o o? ~\ (;) '2 'u(;) ::l tn Graph 8B - Lactobacillus sp. lVfcthylated derivatives. 0'\ ~ 70 .. :r::::-:_. ===== __.:;?_ O!laOV ~ [ ---- 71 u 72 IoqO;)N ---~ ~~~ ... 73 74 ------. 75 c:::::------__~ ______~ 76 o o> I.. e.. TABLE IX Anaerobes and Aerobes Recovered til r:; Cf.l Cranial 2 4 2 2 2 0 Abdolnina1 14 4 2 26 5 6 2 13 19 ]5 Thoracic 7 3 16 6 3 7 10 7 Female Pelvic 5 3 1 3 14 2 2 10 12 . 10 \Vound 8 2 4 17 2 5 9 14 10 .... Genitourinary 2 3 3 :;, 0 Urine 4 1 5 5 5 0 Extremities 6 1 1 12 2 4 2 4 8 6 Peri Rectal 4 1 5 5 5 5 Other 6 1 12 2 3 2 5 8 7 58 16 4 10/88 114 21 33 7 53 86 60 65.9% 18.2% 4.5% 11.4% 18.4% 29.0% 6.1 % 46.5% 75.4% 52.6% ...... TABLE X A. Aerobes Isolated [rOITI Various Sources >. Q ..... ~ f:I:I '> (1) C':! d .S ";3 - .= ...... eo Q Q .§ ~ ;:l (U 13 c:: '0 Il) '"t:) 0 ·s t::: c:a ~ ..... (U ~ .Il) 0 .... t::: .... Il)'"" ";3 "2 '"t:) "'2 ;::l '2 ...... u ~ 0 > ...... ,... Il) (U >< -5 0 ~ U < ~ t:.L. ~ '-' D;.l ~ 0 ~ ~ Number 4 26 16 14 17 8 12 5 12 114 Percentage 3.5 22.8 14.0 12.3 14.9 7.0 10.5 4.4 10.5 Staph aureus 1 2 1 5 6 Staph epidermidis 2' 2 2 8 9 Group D Strep 1 2 3 3 i 1 2 1 3 Group A Strep 1 1 2 3 Other Strep 6 1 2 1 2 2 3 17 19 Proteus species 1 2 1 4 5 E. coli 8 5 3 3 1 1 21 24 Klebsiella/ Entcro bacter 2 1 4 5 PSCUdOlTIOnaS 2 1 3 4 5 Diphtheroids 1 1 Other Gram Positive 1 1 1 2 5 6 Other Gram Negative 2 2 2 23 9 13 15 8 6 13 87 100 26.4 10.3 14.9 17.2 9.2 6.9 14.9 '-oJ 0:> TABLE X (cent.) B. Anaerobes Isolated from Various Sources >. u I-! cd v.I ";j :E CI.) c; CI.) CI.) .S ...... OJ) u t:l-. I-t cd .S ;::::l u ..... CI.) '§ (!.) 2 '0 "0 0 .... ~ cd C; r. .... (!.) ~ (!.) 0 I-t I-< CI.) "a = '2 ;::::l ..... u '"0 0 ·a ..... I-< .c: .... I-! cd ..... E 0 CI.) .... I-< .n (!.) X CI.) 0 CI.) u < E= t.t... ::: c.:> u.J t:l-. 0 f-! t:l-. B. fragilis 12 3 3 4 5 2 1 31 20.1 B. melaninogenicus 1 2 1 1 5 3.2 B.ora1is 1 1 1 1 1 1 6 3.9 B. corrodens 1 1 1 .7 B. species 2 2 3 2 1 11 7.1 Fusobacterium species 2 3 3 1 1 1 1 13 8.4 Propionibacterium acnes 1 4 6 3.9 Propionibacterium species 1 .7 Lacto bacillus species "I 1 4 2.6 Bifido bacteriuln species 3 1 1 6 3.9 Eubacterium species 1 1 2 5 2 11 7.1 Clostridiunl perfringens 1 1 1 4 2.6 Clostridium species 2 1 3 2.0 Veillonella species 1 2 1 1 6 3.9 Pc. assacharo1yticus 1 2 2 2 7 4.6 Pc. prevotii 3 3 6 3.9 Ps. magnus 1 2 5 3 1 1 14 9.1 Ps. intennedius 3 2 2 1 9 5.8 Ps. anaerobius 3 3 1 9 5.8 2 32 14 29 32 14 12 6 14 154 ...... 1.3 20.8 9.1 18.8 20.8 9.1 7.8 3.9 9.1 \.0 80 Table XI indicates the anaerobes that were isolated with aerobes and the number of times that they "/ere isolated together. E. £2.li and i. fraqilis. both normal flora of the intestine. v/ere isolated together most often. Lumping all of the anaerobic gram positive cocci together. they are isolated VJith I. coli about as often as .§.. fragilis. The number of anaerobes isolated together a~ mixed cultures are shown in Table XII. The most common organisms to be found in mixed cul ture \'Ie,"e Peptostrttntococcus ma51tlus and Eubacteri um iQ.. at 11.1% each. They \1ere most often found vii th each other or a speci es of 41 'Bacteroi des not fraqi! 1i s The most commonly isolated anaerobes and their relative frequency are presented in Tab 1e XI I I • §.._ .!r~ ...l..1 ..~. \vas the mas t common VIi th l8~~. TABLE XI Anaerobes and Aerobes Isolated Concurrently Vl I!:n .~ <:> u ....CD TABLE XII Anaerobes Isolated Concurrently With Other Anaerobes ell Q.) ell ell s:: s:: U ell (\) .S C;:1 ell U Co) (\) 00 ell ell (\) t::: ell =' 0.. E E 'u(\) '0 (\) =' u ell ..... (\) ~ ,~ :::: =' 0.. '0 .... '2 .-~ 1-< CI) s:: 0.. (\) ell (\) E ll) ll) ell ""' (\) >-. ell ell ~a 0.. =' 00 ell ...... 0.. ""'""' .~ t) :::l r.n 0 0 u Co) E 1-< ..... :e =' s:: .... C;:1 C;:1 (\) :::: (\) (\) ~ ...... ;:s S S ~ ~ '.;:1 ell :.0 IJ.) ... .n .n u 'i:: ;:s :;j ...... ;:S 0 OIl '2 "'0 'u ~ 0 S 1-< ~ u I;';l (\) v f3 s:: ...... Co) ro ,:S 0 '2 '2 C;:1 ~ ...... 1-< ro ..0 :§ :e s:: (\)> 00 (\) '5h ...... 0 0 ..0 CI) ro ...... ro c'j CJ ~ ..0 0 U 1-< 'i:: ...... s:: ...... 0 ro ...... 0 en s:: (\) 0 0 '0...... "'0 en ell ro 0.. s:: ...:: S 0 U en 's. u ..0 ~ S ro "a.... u ;:s 0 0 ~ 0 0 .- 1-< 1-1 :.... :::l u "..; 0 C!i ~ p:i ~ cQ u.. A-I ~ j 20 ~ U U ~ A-I ~ ~ A-I ~ r:-. &:: B. fragilis 2 3 1 3 1 1 1 1 1 18 7.1 B. melaninogenicus 1 2 1 1 3 1 2 1 2 2 17 6.7 B.oralis 2 1 1 1 1 1 1 1 12 4.7 B. corrodens 1 1 1 6 2.3 B. species 2 1 1 2 1 2 1 6 2 1 23 9.1 Fusobacterium species 2 1 1 2 2 15 5.9 Propionibacterium acnes 1 2 7 2.7 Propionibacteritl1n species 1 1 4 1.5 Lactobacillus species 1 1 1 1 10 3.9 Bifido bacterium species 2 2 1 1 14 5,5 Eubacterium species 1 3 2 4 3 5 28 11.1 Clostridium perfringens 0 0 Clostridium species 2 .7 Veillonella species 1 8 3.1 Pc. assacharolyticus 2 2 2 1 17 6.7 Pc. prevotii 1 1 9 3.5 Ps, magnus 3 1 28 11.1 Ps. intermedius 1 16 6.3 Ps. anaero bius 17 6.7 co N TOTAL 251 83 TABLE XIII The Most Frequent Anaerobic Isolates Bacteroides fragilis 18% PeptostreptococCU5 magnus 8% Fusobacterium species 8% Eubacterium species 7% Bacteroides species 7% Peptostreptococcus intermedius 6% Peptostreptococcus anaerobius 6% B. oralis 5% P. acnes 5% Bifidobacterium 5% Veil10nella 5% Pc. assacharolyticus 5% Pc. prevoti; 5% v DISCUSSION Present literature is limited in the amount of information in ansver to the questions posed at the beginning of the paper. Some of the reasons are: 1. The procedures used to gather the information were open to question. Either the collection methods were not adequate to ensure that all clinically signific~lt anaerobes would survive the tri p to the 1aboratory or the i so 1ati on methods "tcre not adequate to recover these organisms. fulother reason is that the procedur'es that viera used to i dent; fy the organ; sms vias open to question since in some reports all the gram negative rods Nerestill called "bacteroides". 2. The second reason is that the data was based on a specific group of patients such as predi sposed patients VI; th varying underlying diseases~ 3. Some have reported only the number of each of the species that have been reported to them. This gives no indication as to the numbers of organisms isolated but rather the number of problems that others had in speciating them. 4. The organisms reported are not consistent with other reported data. For example. Zabransky (1970) did not report any ~. melaninoqen;cus, 1:. ~~. or E.ubacterium. all of \ After setti n9 up the tV/O anaerobi c systems, we found several advantages and disadvantages of each. Glove box Advantages 1. Can gri nd up ti ssues for cu1 ture vIi thout hei ng exposed to ai r. 2. Can use routine media and reduce it in the glove box thus cutting the cost of media. 3. The cost of operating is nominal. 4. tHnir:lal training required. Disadvantages 1. Uncomfortable to work in because of the heat. After using it for a fe\" mi n utes, your hands and arms are vIet vIi th persp; ra tion hecause of the plastic gloves and sleeves. 2. Time consuming. The time required to enter a speciMen varied but took at least ten minutes. 3. The gloves are difficult to work with while handling plates and equipment. 4.· Initial cost is very high. 5. Large amount of space required. 86 6. Difficult to see the colonies on the plates because of the plastic walls of the cham~er and the lighting. E.oll tubes Advantages 1. Easy to inoculate individual specimens as they come in with a minimum of time. 2. Tubes can be observed at any time and shovm to several different people without putting their hands into the glove ports. 3. The space required is minimal. 4. Moderate initial cost. Disadvantages 1. Commercially prepal"ed media expensive. Pr'eparation of the roll tubes is time consuming and requires extra equipment and training for the media maker. 2. Colonial morphology frequently is not distinctive because of the requirement of transparent media. 3, Requi res practi ce to be able to roll a tube \'Je 11. Tili sis not the problem that it first appears to be. 4. Ho selective media is conrlercia11y available. After the gas chromatograph \Vas set up and running, \:Ie had very few problems with it. There were two exceptions that should be noted. It was found that the disposable plastic tubes that we had intended to use for the extraction procedure were soluble in ether and that after injecting this ether extract of plastic into the column, the gas chromatograph ceased to function in a predictable manner. The 87 plastic condenses on the exit ports and plugs them up causing a change in the flo':1 rates and sharp peaks on the pattern to The second problem that was encountered was that of an extra peak eluting off the column after the succinic acid peak. This was not expected since no other peaks are reported by those that have done \'/ork vii th anaerobes. A copy of the gas chromatograph pattern along with the identification of the organism was sent to Dr. W. E. c. Hoare. He stated that perhaps, it vias a carryover of the volatile products and that a correspond; n9 peak \A/oul d be found on the ether extracted sample of the sarne organ; sm. None \'1as found hm'Jever and the identification of this peak still remains to be determined. This peak di d not ; nterfere \lJi th the i dent; fi cat; on hov/ever. Because of our inexperience. the orqanisms that were isolated and identified at the first part of the study were misidentified occasionally. After a period of time the identification charts of these original organisms were looked at in the light of a little experience and found that some \'/ere most likely speciated VJrong. Hm'Jever, to go back and try to change the identification \ Because the co 11 ecti on procedurt~ \lIas unfami 1i ar to all of the house staff and others collecting the specimens many were not collected properly and we had no way to evaluate this. Another point that must be considered \'Ihen evaluating the data. is that only specimens that were specifically ordered for anaerobes were 88 done. This was not set up as' a routine procedure and the routine specimens \'/ere not handled any di fferently than they had before. Because of the expense of the culture ($25.00). doctors would only order anaerobic cultures if they suspected an anaerobic infection. Thi s v/oul d tend to make the percent recovery much hi gher than normal. The 75% reported here is probably higher than would be expected if more specimens were cultured. The data shown here point out that it is important to isolate anaerobes in culture. You cannot rely on gram stains of either the original speci8en or thioglycollate broth to detect their presence. It is true that if you see an organism on gram stain and it fails to gro\,l. that an anaerobe should be suspected. Hov/ever. in many cases they are mixed cultures of aerobes and anaerobes. Since the most commonly found organisms in each group are gran negative rods. the anaerobe would likely be overlooked unless the technologist has a great deal of experience readin9 these types of gram stains. Because the identification as to genus and species of most of the clinically important anaerobes may be academic at the ptesent time and usua lly \,/i 11 not change the recommended therapy except in the case of B. franilis or certain Clostridia, it is questionable if the __ '4 ""1'IiI." identification of all the anaerobes is necessary. Usually only ~. fragi1is requires a chanaes in therapy. Speciation is a good epidemiological tool and can be helpful in sor!1e cases. He are not presently speciating all of the anaerobes isolated, only those that are of clinical interest or those that will alter the antibiotic regimen. We have considered screening all specimens for B. fragilis 89 and i denti fyi ng a 11 others to genus on 1y •. Except i cns \'loul d be those in the genus C)ostridum and isolates from blood and other areas that are nonna11y considered to be sterile. ~Je found that gas chromatograph to be very helpful for identifying anaerobes. There are other \ To improve the performance of the gas chromatograph we doubled the f1m'l rate of helium from 120 cc per minute to 240 cc per minute. There is also a nevI packing available from Dohrmann Envirotech \I/hich 'vIC have started to use. Between these b'lO changes \'Ie \'Iere able to bring the elution times dONn to about one third what they v/ere in this paper. The procedures used in this study are not necessarily recommended fO)'" routine usc in clinical laboratories. The glove box was found to be ve)"'y cumbersome and much aversion Has developed to its use by the microbiology staff. Of the two systems used here. the roll tube is most aop1icab1e for routine use. It has been sh~/n recently by several investigators that conventional media, used on the bench. without the advantages of a system to prevent exposure to air. and keeping the exposure time to a minimum. is adequate to isolate most of the currently considered clinically significant anaerobes. Hmvever, it remains to be proven which anaerobes are clinically significant. 90 The qUestion of \'lhich anaerobes are clinically significant is one that remains and still is asked many times. Another question that arises ;s how far should the laboratory go in speciating the various organisms presented to it? The expense must be considered. also the clinical usefulness of the data to be evaluated. Are we adding confusion by reporting all the various organisms or is that data helpful to the physician? The feeling at the present time is that the only organisms that will change the patientls therapy are !!.. fraqilis and certain species of c·lostridia. Another" 'interesting question that has come up is, ho\'! well can the average technologist. \'lith some training, read the gram stained smears of the specimen and determine if anaerobes are present or not. There has been the feeling that the corr'elation is very good. If that is true then the gram stain of the original specimen can be very helpful for those laboratories that are limited in their ability to isolate and identify anaerobes becuase of lack of anaerobe jars and other equipment. There are many questions that need to be answered before every one agrees on the role of the anaerobes in clinical laboratories. The general feeling at the present time is that He must at least be able to isolate anaerobes from specimens and tell the physician that thOey are present and their gram morphology. This means that the collect.ion procedures must be improved and the laboratory must be aV/are of "Jhat specimens contai n anaerobes and how to recover them. BIBLIOGRAPHY Aranki. 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HcIntosh. \J.; Fildes, P.. 1916 0 A ilet'J l'~ethod for the Isolation and Cultivation of Anaerobic f1icl"oorr~anisms. lancet 1916:768. ~klntosh J J.; Fi 1dBS J P. 1921. An Improved Form of fklntosh and Fildes Anaerobic Jar. Brit. J. Expt. rathol. 2:153-154. r'1c~1inn. n.T. 1970. Recovery of P.naer·obic Hicroorganisms from C1 ; ni ca 1 Spec; mens in Pre Reduced 1'·1edi a Versus Recovery by Routine Clinical Laboratory i'lethods. App1. r'iicrobio1. 19:207-213. 94 Mitruka. B.M. 1970. Differentiation of Brucella canis from Other Bruce 11 ae by Gas Chror.1a tography. App 1. r1i crobi 01. 20: 649-650. t4oore, H.E.C.; Cato, Elizabeth P.; Holdeman, Lillian V. 1966. Fermen tation Patterns of Some Clostridium Species. Int. J. Syst. Bact. 16: 383-415. f1oore, \~.E.C.; Cato, Elizabeth P.; Holdeman, Lillian V. 1969. Anaerobic Bacteria of the Gastrointestinal Flora and Their Occurrence in Clinical Infections. J. Inf. Dis. '19:641-649. Parker, e.A. 1955. Anaerobiosis with Iron Wool. Austral. J. Exp. r~d. Sci. 33:33 .. 37. Porter, Ruth (Ed.) 1969. Gas Chromatography in Biology and !Aedicine. A CInA Foundation Symposium. J &A Churchill. Ltd., 104 Gloucester Place, London, England. Rose, S.B. 1942. The Importance of CO2 in Diagnostic Bacteriology with Observations on the CO 2 (capneic) Incubator. Am. J. elin. Path. 12:424-433. Rosebury, T.; Reynolds, J.B. 1964. Continuous Anaerobiosis for Cultivation of Spirochetes. Proc. Soc. Exp. Biol. Med. 117:813-8150 Rosenblatt, J.E.; Fallon, A.; Finegold. S.M. 1973. Comparisons of f'lethods for" Isolation at' Anaer'obic Bllcteria from Clinical Specir:lens. Appl. Microbial. 25:77-85. Smillie, H.G. 1917. NevI Anaerobic t-1ethods. J. Exp. i"1ed. 1917:59. Smith, L.D.S.; Holdeman, L.V. 1970. The Pathogenic Anaerobic Bacteria. Charles C. Thomas, Publisher, Springfield, Illinois. Society of J\merican Bacteriologists. 1957. (:tcGrm" Hill, r~e~1 York. Sutter. Vera L.; et~. 1972. Anaerobi c Bacteri 01 ogy [':1anua 1. Depart ment of Continuing Education in Health Sciences. University of 95 Extens; on and the Schoo 1 of f1edi c; ne t UCLA. Spray. R.S. 1930. An Improved Anaerobic Culture Dish. J. Lab. C1in. r·1ed. 16: 203-206. Spray. R.S. 1936. Semi ... so1id f,1edia for Cultivation and Identification of the Sporulating Anerobes. J. Bact. 32:135. Wilson. G.S.; Miles, A.A. 1964. Tapley and Wilson's Principles of Bacteriology and Immunity. Vol, I. p. 83. The L-1il1iams & t~filkins Co.. Ba 1ti r.lOre. r1a ryl and jj Zabransky. R.J. 1970. Isolation of Anaerobic Bacteria from Clinical Specimens, Mayo C1in. Proc. 45:256-264. VITA Name James Ira f1iller Birthplace los Angeles. California B1rthdate r·1arch 15, 1942 ~1arried linda lou Loveland September 10, 1966 Children Darcine February 7, 1968 Stephanie f4ay 30 t 1969 Allison April 22, 1972 f1eri 1ee January 7 t 1974 High School Franklin High School Los Angeles, California College Pasadena City Colleqe 1960-1961 Pasadena, California College East Los Angeles College 1964 Los Angeles. California UnivGrsity Brigham Young UniVC1"sity 1965-1967 Provo, Utah Degree B.S. s Brigham Young University 1967 Provo, Utah Medical Technology Saint Benedict's Hospital Internshi p Ogden. Utah 1969-1970 Universi ty University of Utah • 1970 ... 1974 Salt Lake City, Utah Certificate American Society of Clinical Pathologists t1edi cal Techn 01 ogy Reg; s try Saint Benedict'sHospital Ogden, Utah Professional Organizations American Society of Clinical Pathologists Ameri can Soc; ety of lied; ca 1 Techno 1ogi s ts American Society of l'1icrobiology 97 Vita (Continued) James Ira Miller Professional Positions Labol"atory f'1anager- t Department of Microbiology, Weber State College, Ogden, Utah; f,1edical Technologist Consultant. Intermountain Regional r·1edi ca 1 Program, Un; vers ; ty of Utah, Salt Lake City, Utah; Supervisor. C1 i ni ca 1 f,1i crobi 01 oqy , Uni vers i ty of Utah t'1edical Center, Salt Lake City. Utah Abstracts Nosocomial infections and bacteriology laboratory services in small cOMmunity hospitals, Britt, f1.R., r~ordquist, A.G Il • Miller. J.I., Hi1fert, lJ.N., Bur'ke. J • .,]. International Congress of Antimicrobial Agents and Chemotherapy 19738 Quality assessment of bacteriology laboratories using prevalence survey techniques. Miller, J.I., Britt, M.n. Intet"Plotmta-j n Branch i\mer; can Soc; ety for Hicrobio109Y 1974.