Suggestions on Bacteriological Techniques for the Beginner L

Home , Inoculation loop, Inoculation needle

Suggestions on Bacteriological Techniques For the Beginner L. S. McCLUNG Departmentof Bacteriology, Indiana University,Bloomington

For those who have not had training in bac- possess this, microscopes so equipped may teriology, casual visits to the laboratories in often be borrowed from a local physician, which extensive work is being done might in- hospital, or industrial laboratory. dicate that the techniques necessary to the Many of the techniques discussed in this various procedures are difficult to master. article are illustrated in the films, Bacteria: Downloaded from http://online.ucpress.edu/abt/article-pdf/22/6/343/16662/4439362.pdf by guest on 29 September 2021 Such is not the case, and in this article a few Laboratory Study and Elementary Techniques pointers will be given to guide the beginner. in Bacteriology. Abstracts of these films are A good rule to remember is that "Practice included in the microbiology Teaching Aid Makes Perfect." Do not expect to be proficient Lists on page 370 in this issue. the first time. Many valuable suggestions, some of which Use of Microscopic Preparations will be repeated or expanded in this article, For detailed examination of microorganisms will be found in A. C. Lonert's, Turtox Bac- in the living condition, microscopic prepara- teriology Booklet, An Introduction to Bac- tions of two types may be made. For the wet teriological Methods, published in 1946, and mount, all that will be needed will be clean sold, $1.00, by General Biological Supply slides and cover slips. These may be used re- House, 8200 South Hoyne Avenue, Chicago peatedly if they are thoroughly washed after 20, Illinois. For additional material, the fol- use with Bon Ami or a similar cleaning agent. lowing books are recommended: Slides should be sufficiently clean that a drop Conn, H. J. (Ed.) Manual of Microbiologi- of water will spread in a thin film instead of cal Methods, McGraw-Hill Book Com- remaining as a drop, and frequently new slides pany, Inc., New York, 1957. are covered with a film of oil which must be Skerman, V. B. D. A Guide to the Identifi- removed before use. To prepare a wet mount, cation of the Genera of Bacteria, Williams use pipette tip or inoculation loop to place a and Wilkins Company, Baltimore 2, Md., large drop of the liquid culture or infusion, or 1959. make dilute suspension of colony material in Tanner, F. W. Practical Bacteriology: An drop of tap water, and carefully lower cover Introduction to Bacteriological Technic, slip in such a manner that no air bubbles are 2nd ed., John Wiley and Sons, Inc., New trapped underneath it. If the drop is too small York, 1933. for a coverslip, air bubbles are almost inevi- These books, which contain valuable sugges- table, but the amount of liquid should not be tions on formulae of media, preparation of so great that the cover slip appears to float. In staining solutions, staining techniques, etc., this event absorb excess liquid with pieces of assume that the user has had some training in blotter. bacteriology. With the suggestions given be- A second type of wet preparation, a hang- low, they should, however, be very valuable ing drop suspension, employs a concavity to the beginner. slide, usually of same thickness as ordinary Although microscopic examination of bac- slide, and coverslip. Place a ring of petrolatum teria provides much valuable information, it (vaseline) around the edge of the concavity should be remembered that many experiments and add coverslip on which drop has been may be done involving cultivation of bacteria placed in such a manner that drop hangs sus- which do not involve the use of a microscope. pended in concavity. The wet mount will If microscopic examinations are attempted, ordinarily remain satisfactory for examina- the microscope should be equipped with an tion for 10-15 minutes; the hanging drop, pro- oil immersion objective. If the school does not tected from evaporation, will last for a longer 343 344 THE AMERICAN BIOLOGY TEACHER June, 1960

period. To examine either of these prepara- oil immersion objective, the characteristic tions, use the low or high power objective of shape and arrangement of bacterial cells. An- the microscope. The usual failure to obtain an other method of examination, called the nega- image results from too much illumination; if tive stain, is also useful for the same purpose. microscope is provided with a diaphragm this For this add a loopful of black or blue India should be partly closed. The beginner might ink to the drop of culture on a slide before practice with 48 hour old hay or pepper in- drying and spread out in a thin film. Upon fusions until success is attained before attempt- examination, the bacterial cell will appear un- ing to study the more dilute bacterial cultures. stained, clearly outlined against the dark For preparation of stained smears, use an in- background. oculation loop to spread a thin film of liquid The Gram stain is a differential stain often culture in an area about one-half inch in di- used in bacteriology. The staining technique, Downloaded from http://online.ucpress.edu/abt/article-pdf/22/6/343/16662/4439362.pdf by guest on 29 September 2021 ameter on the clean slide. If colony material which is given in the books listed above and from agar culture is to be examined, place a on page 304 of this issue, employs alkaline drop of tap water (finger tip is satisfactory for crystal violet as the primary stain with iodine this purpose) on slide and, with the inocula- as a mordant. Following decolorization with tion needle, make dilute (barely touch colony) alcohol, the counter stain of safranin is ap- suspension. Use a tooth pick or wooden swab plied. Those cells retaining the crystal violet stick if the inoculation needle and loop are not are called Gram positive, and those which lose available. Allow material smeared on slide to the violet during decolorization and take up dry in air (2-3 minutes is usually sufficient) the red stain are called Gram negative. The and heat-fix to slide by passing slide (smear Gram reaction of a .species is of taxonomic side up) through bunsen or alcohol lamp flame importance and also correlates with other two or three times. Such a preparation must properties. For example, Gram positive or- be stained for best examination. ganisms are more susceptible to the action of The books listed above and most introduc- penicillin than Gram negative types. For cor- tory textbooks of bacteriology will give direc- rect results the culture should be not more tions for a variety of staining procedures, but than 24 hours old, if it will grow within that many substitutions may be made. Gentian or period, and the smear should be thin. The crystal violet is often available at the drug critical step is the decolorization with alcohol. store and is recommended for this purpose. For examination of stained smears, place Make a solution of 2 grams of the dye dis- slide on stage of microscope, and, turning solved in 20 ml. of alcohol (iso-propyl or rub- head to side, carefully lower oil immersion ob- bing alcohol is satisfactory) and dilute this jective until it touches drop of oil which has with 80 ml. of water. Use 5 ml. of this con- been placed on top of stained smear. Then, centrated solution (which will keep for long using more illumination than for wet mounts, periods) and 95 ml. of water to make the final focus upwards slowly until image is obtained. staining solution. To use as a stain, add (us- The usual fault with smear preparations is that ually from dropper bottle) the stain solution the smear is too thick. This is ordinarily not directly to smear, and let stand at room tem- true of mounts from liquids, but suspensions perature for 1-2 minutes. Wash slide with prepared from colonies should not be "milky" gentle stream of tap water, or use plastic or before drying. Bacterial cells will be stained glass chemistry wash bottle, or "dunk" slide solidly by this procedure, but spores will be in tumbler of water. Hold slide with blunt stained in outline only and center xvill be clear tipped forceps to avoid staining fingers. (Figure 1). If supply funds do not permit purchase of The oil must be removed from tip of objec- dyes for staining procedures, try colored inks tive each day before the microscope is placed as a substitute. The permanent purple inks, in in storage cabinet. Use lens paper for this, and our tests, have been the most satisfactory. For if oil is sticky use a small (to prevent dissolv- these the time of application may need to be ing lens cement) amount of xylol on lens extended to 5-10 minutes. paper. A satisfactory substitute (water is not) The simple staining solutions mentioned for the cedar oil commonly supplied for this above will reveal, upon examination with the purpose may be made by adding 28.0 ml. of Vol. 22, No. 6 SUGGESTIONS ON BACTERIOLOGICAL TECHNIQUES 345

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Figure 3. Method of holding tubes, plugs, and inoculation needle during transfer of cultures.

should be included concerning use of them. The wire of the inoculation loop or needle should always be heated to redness in a bunsen flame before and after use (Figure 2). It may be cooled by holding momentarily in air or, for agar cultures, by touching spot of agar where there are no colonies. To transfer from Figure 1. Vegetative cells and spores of rod one tube to another, hold tubes as if they are shapedbacterium. extension of fingers in one hand, and with the other hand which holds the inoculation loop remove cotton plugs by holding between the alpha bromnaphthalene to 100 ml. of light fingers with the back side of the hand (Figure weight mineral oil. This oil does not become 3). Sterilize mouth of tubes or other con- sticky and is very satisfactory for routine use. tainers by passing through a bunsen flame after plugs are removed. For pipettes (usually Transfer of Cultures a small plug of cotton is inserted in mouth end Fagle and McClung (1960) have described a and flamed so that no cotton protrudes be- variety of devices for inoculating bacterial yond tip), use the forefinger (not the thumb) culture solutions, but perhaps a few pointers to control the level of liquid (Figures 4 and 5). After use, pipettes should be discarded in a tall jar or long tray of disinfectant.

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Figure 4. Pipetting technique for aseptic transfer Figure 2. Flaming of inoculation loop. of liquids. Note bottle cap held between fingers. 346 THE AMERICAN BIOLOGY TEACHER June, 1960

be plugged with cotton before sterilizatiotn. Pipettes should be sterilized in long metal cans made especially for this purpose. Acceptable substitutes include cylindrical cardboard mail- ing tubes plugged with cotton at both ends. Pipettes may also be wrapped in groups in paper or metal foil. Special single service, thin, paper tubes have recently become available for this purpose. A supply of sterile glass petri dishes is es- sential for many experiments in bacteriology

unless pre-sterilized single service plastic plates Downloaded from http://online.ucpress.edu/abt/article-pdf/22/6/343/16662/4439362.pdf by guest on 29 September 2021 are employed. To avoid the necessity of ster- Figure 5. Delivery of liquid to petri plate by ilization of the dishes immediately prior to use, pipette. For photographic purposes the plate, it is recommended that the plates be sterilized which should remain on table, is being held in cans such as one pound coffee cans or after towards camera to show method of holding wrapping in newspaper with scotch tape to cover. make packet units secure, in unit packages of four to six plates. This affords protection against dust contamination after sterilization. Preparation and Sterilization of Glassware Although other plates are satisfactory, the For most laboratory experiments the dry 15 x 100 mm. assay plate with thick glass and glassware should be sterilized before use, flat bottom, available as Pyrex #3162 from t'hough in many short term experiments abso- most supply houses, is recommended if a new lute sterility is not necessary. For all experi- supply is to be purchased. These are used with ments, however, the glassware should be very glass (Pyrex #3160) or metal tops (consult clean. After use and before washing, glassware catalogue of Baltimore Biological Labora- containing cultures should be sterilized either tory) (Figure 6). Petri dishes are useful in in steam (autoclave or pressure cooker) heat dilution plate experiments for enumeration of or thoroughly boiled in water. The glassware bacteria in soil, water, milk, foods, etc., for should then be washed using any good house- pure culture isolation, for many experiments hold soap or detergent and then be thoroughly on effects of environment on bacteria, and for rinsed. Good rinsing is particularly important many other purposes. if detergents are used, for otherwise a thin film of detergent nmayinhibit bacterial growth when the glassware is subsequently used. It is .N better to dry the glassware by rapid evaporation (place in oven at low heat or in some other area where the rinse water will evapor- ate quickly) rather than wiping with cloth. Although sterilization of dry glassware is not absolutely required for tubes or flasks which are to receive media to be sterilized bv steam heat, presterilization of the dry glass- Figure..6..Glas.ndmealtopedperidihe ware will reduce the load of bacterial contamination to be killed when the medium is sterilized. Thus this is recommended particularly when there may be some doubts concerning the adequacy of the sterilization process for the media. This sterilization is usually accomplished by heating in an oven (cook stove with regulator is satisfactory) for at Purchase of Bacteriological Media least one, and preferably two, hours at 1800C. Culture media (singular, medium) for bac- or 3560F. The necks of tubes and flasks should teria are of various types, and the choice will Vol. 22, No. 6 SUGGESTIONS ON BACTERIOLOGICAL TECHNIQUES 347 depend on the purpose intended. Some are used simply to grow cultures for microscopic 0 ';:XT;ttiEl:,' :' t:ft; > x' -: examination or other purposes while others ';: ...... ' .f.':.'.S.'.'.. '"""{'|..S...... SS. will reveal certain metabolic patterns following growth. Space will not permit a full dis- '''.{.';'..,.,,....,',S S1,,,,,,,.,..,,,,i,......

.'.'.'.'." SS cussion of this topic, and those who need ad- .....i S t iE ditional information should refer to Chapters ' ' S ' ,~~~~~~~~~~~~~~~~~~~~~~~~~... , i...... s'.' W ... g X ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~....s,>..... II and VII of the Manual of Microbiological Methods and to the section of methods in the books by Skerman or Tanner.

The most convenient way of preparing many Fiur 7. Reovn bakt of tube mei fro.s;m auto lav .ss t of the media used for cultivation of bacteria Downloaded from http://online.ucpress.edu/abt/article-pdf/22/6/343/16662/4439362.pdf by guest on 29 September 2021 is to use the dehydrated media prepared by one of the commercial compaines. The two largest companies which prepare media are Difco Laboratories, 920 Henry Street, Detroit 1, Michigan, and Baltimore Biological Labo- ratory, 2201 Aisquith Street, Baltimore 18, Ave., Chicago 20, Illinois. For media ingredi- Maryland. Products of both of these com- ents, usually one-quarter pound quantities are panies are more easily obtained from one of sufficient for experiments for one year. Agar, the general supply houses rather than the com- used in 1.5 -2.0% concentration for solidifica- pany directly. As indicated elsewhere, both of tion of nutritive media, may often be pur- these companies will send on request compli- chased in local drug stores. mentary copies of catalogues and price lists. To use dehydrated media, the correct Preparation of Media amount, usually indicated on the bottle, of the For routine cultivation of many bacteria dehydrated powder is added to water, the two very useful media are nutrient broth powder dissolved by heat, and dispensed to (liquid) and nutrient agar (liquefiable solid). appropriate tubes, flasks, or bottles, and steri- The formula for nutrient broth includes 0.5%l lized. The cost of dehydrated media is greater peptone and 0.3% beef extract. The appropri- than the cost of the ingredients which may be ate amounts of the dry peptone powder and purchased separately, but, in addition to the the sticky beef extract are weighed out and convenience of time saving, these products dissolved in tap water. Distilled water is often offer the advantage of uniformity and thus listed for this medium but is unnecessary. The are recommended when funds permit. Simi- ingredients are dissolved by gentle heat and larly, the companies mentioned and others the pH adjusted to 7.0 (by addition of IN prepare pre-sterilized media in screw cap bot- HCI or NaOH) if colorimetric pH indicators tles or tubes, and while these are still more indicate necessity of change. If tested with expensive, they may be preferred in labora- brom-thymol-blue, the indicator should be tories without sterilization facilities. grass green in color. If agar is desired, add 1.5 Other companies which sell media ingredi- to 2.0 grams per 100 ml. of medium. ents or pre-sterilized media include Albimi If peptone and beef extract are not avail- Laboratories, 16 Clinton Street, Brooklyn 2, able, do not abandon attempts to cultivate N. Y.; Case Laboratories, Inc., 515 North Hal- bacteria for satisfactory substitutes are easily sted Street, Chicago 22, Illinois; Hyland Lab- available! Try one of the formulae suggested oratories, 4501 Colorado Blvd., P. 0. Box by Sister Paulinus (1960) or one of your own 39672, Los Angeles 39, California; and Media, from materials available to you. Additional Inc., 89 Lincoln Park, Newark 2, New Jersey. suggestions: (1) make boiled infusion of 250 As indicated above, most of the general sup- grams of hamburger in a liter of water, filter ply houses sell the products of these companies to remove meat particles, cool and remove fat, as do certain of the biological supply houses, adjust pH. (2) Add 250 grams of rich soil to such as Carolina Biology Supply Co., Elon one liter of water, boil gently, strain, filter,- College, North Carolina; or General Biologi- and adjust pH if necessary. (3) Add 5 grams. cal Supply House, Inc., 8200 South Hoyne of yeast cake to 500 ml of water, boil gently, 348 THE AMERICAN BIOLOGY TEACHER June, 1960 filter, and adjust pH if necessary. (4) Steep periments the exact control of temperature is 100 grams of hay or dried grass in one liter of not critical. Here is another project for in- water, filter, and adjust pH. The use of these genious students who have shop facilities avail- as the nutritive base medium with addition of able. agar should, with simple air exposure plates, Agar plates should be inverted agar side convince the most skeptical that bacteria can up during incubation. This will prevent the be cultivated on inexpensive materials. water of syneresis from forming a thin film on the surface which would permit bacteria Sterilization of Media to grow in a confluent film rather than form- Most of the media used in bacteriology ing discrete colonies. should be sterilized either in a steam autoclave as shown in Figure 7 or in a pressure Pure CultureIsolation Techniques Downloaded from http://online.ucpress.edu/abt/article-pdf/22/6/343/16662/4439362.pdf by guest on 29 September 2021 cooker. The time required is usually 15-20 Often it is desired to isolate a pure culture minutes after the pressure reaches 15 pounds of a bacterial species from the mixed popula- and the temperature should be about 2400F. tion of a natural sample. The material to be A cardinal rule to remember is to let the pres- studied may be a liquid culture or a suspen- sure in the autoclave or cooker return to zero sion in sterile tap water of colony growth before removing the sterilized media. To from an agar culture. A pure culture refers force the pressure down, by opening the steam to a culture containing the cells of only one escape valve, will cause the superheated media species. Two methods for obtaining a pure to boil over. Never shake or swirl a flask or culture are recommended and the first is pre- bottle of agar media immediately upon re- ferred since less glassware is involved, and it moval from sterilizer as this will also cause the has other advantages. medium to boil over and the burn is very pain- Method 1: Pour nutrient agar plates and ful. The media must be sterilized the same day allow medium to harden. It will be better if that it is prepared, preferably within 2 hours. Storage of Media The general rule, unless screw capped tubes _ ~~~~~~~~~~~~~~~~~~~~...... or bottles are employed as the container, re- _ -__ _ _ ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~.. .+...... garding media storage, is to make and sterilize only the media needed within the next week or ten days. Longer storage will bring evaporation problems unless refrigeration ture| tube., ...... space is available. Prolonged storage under refrigeration sometimes involves contamination by mites, which travel through cotton plugs, or molds. Media protected from evap- |~~~~~~~~.,.''0 ~ ~~~~~~~~~~~~~~~~~. ...0... . oration by tightly closed screw caps will last practically indefinitely. For these items us- | ~~~~~~~~~~~~~~~~~.*...... 2>....:.L.'-.:. 0:.-....t- .... _ j 9 ' 9 | 0t'-i~~~~~~~~~~..0 '-' ;0i; 00 ...0 . ually the screw cap should be loosened one- .XFgr8. ,'''-.'.,.'',.',....''"''''' Drinn ...... incuato .... loopgwithin<, cul - quarter turn before sterilization and tightened afterwards. ..,.,.,,.,,.,,,..,.,,,,,..-,,,,.,~~ ...... t~ ~ ~ ~~~~...... '' Incubationof Bacterial Cultures '',..,.,...'...... ,.'""-"'''...... C000 Many bacteria, certainly all that would be needed in elementary biology, grow well at room temperature (28-30? C) within ordinary Figure 8. Draining inoculation loop cul-~~~~~~~~~~~~~~~~~...... within ...... 48-72 hours. For some experiments the direc- ture~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~...... ube..... tions may suggest incubation at 370C but often a substitution of organisms may avoid the necessity of provision of this special incubation temperature. The list of reference articles, however, includes suggestions on adapting old refrigerators for this purpose. For many ex- Vol. 22, No. 6 SUGGESTIONS ON BACTERIOLOGICAL TECHNIQUES 349 the plates are allowed to dry for a small number of hours after pouring. They should be inverted during this time. Charge the inoculation loop by dipping in liquid culture or suspension and drain out the liquid from the loop by touching inside of culture tube above the level of the liquid (Figure 8). Then make parallel streaks, without cutting into agar, across about one third of agar surface (Figure 9; in this photograph the petri dish lid has been raised more than it should be to permit

photograph to show relation of loop to agar Downloaded from http://online.ucpress.edu/abt/article-pdf/22/6/343/16662/4439362.pdf by guest on 29 September 2021 surface). Flame the loop, cool it by touching agar, and repeat streaks over one half of re- | maining area, taking care to streak across a Sx~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~.+^...... -. #| portion of the area first inoculated. Flame needle again and repeat process with remaining area on plate. Isolated colonies should appear on one of the sectors following incubation. Such colonies should be emulsified in sterile tap water and a new plate streaked and this, on incubation, should produce colonies of uniform morphology. Method 2: Using tubes or bottles of size to contain about 20 ml. of medium, dispense, and sterilize several such tubes. When needed, liquefy the agar by placing in pan of water Figure 9. Technique for streaking agar plate. The which is heated to boiling point. Allow three plate cover is ordinarily held closer to bottom tubes per culture to be studied to cool in a pan of dish. of water to 40-42?C. Use sterilized inoculation loop to transfer one loopful from culture suspension to tube 1 while agar is still liquid. exercises or special projects as Sister Paulinus Flame needle and lay it on desk. Rotate in- (1960) has suggested. oculated tube between palms of hands and transfer two loopsful from it to tube 2. Repeat How to Identify Bacteria process given above, transferring one loopful At the outset it should be stated that there from tube 2 to tube 3. Before the agar column are no "How to Know Bacteria" books of the cools and solidifies, pour contents of each tube, same type that exist for some larger plants and after flaming tube lip, into a separate sterile animals. Bacteria may not be identified by petri dish. Upon incubation both surface and morphological features alone, and the kzys for subsurface colonies should appear. These may taxonomic purposes appear at first glance to appear to be different, even with a pure cul- be a bit complicated for the beginner. As a ture, for the subsurface colonies are usually matter of fact, they are complicated for the either lens shaped or irregular, wooley balls, advanced student in bacterial taxonomy who whereas the morphology of surface colonies may wish to consider all the possibilities in can be quite varied with different species. tracing the identity of a newly isolated culture. The novice is not likely, however, to need to From the plates prepared by these methods, consider every previously described species! pure cultures may be established by transfer- With this thought in mind a few suggestions ring from an isolated colony to a tube of will be offered which may be of some aid to broth or agar slant appropriate to the type some, though most certainly not all, taxo- being studied. By these methods it is not diffi- nomic problems relating to bacteria. For cult to isolate, as needed, bacteria for class advice on other microorganisms, reference 350 THE AMERICAN BIOLOGY TEACHER June, 1960 should be made to some of the titles in the sample in order that the colony count attained Reference Book List. will lie between 30 and 300 per plate. The "standard" American book on the clas- Tap water is satisfactory for the diluting sification of bacteria is Bergey's Manual of De- fluid, and the appropriate amount should be terminative Bacteriology of which the 7th edi- sterilized in tubes or screw capped bottles. tion was published in 1957 by Williams and The size of the initial sample depends on the Wilkins. Skerman has presented a very useful nature of the sample. For milk or other fluids key which will lead you to the genera in in which the population can be assumed to be Bergey's Manual, but the key necessarily in- evenly distributed, the initial sample of 1.0 cludes many more groups than need to be con- ml. is usually added to a 9.0 ml. or 99 ml. sidered. Most of the types which the beginner dilution blank. With soil or other samples that

will isolate from water, soil, air, etc., will are not homogenous, the initial sample would Downloaded from http://online.ucpress.edu/abt/article-pdf/22/6/343/16662/4439362.pdf by guest on 29 September 2021 probably be included in a small list. The fol- usually be larger. A good example would be lowing list of genera of the bacteria most to add 10 gr. of soil to a 90 ml. dilution blank likely to be isolated may be used as a prelimi- or 50 gr. to 450 ml. For the purposes of these nary guide to more detailed descriptions in samplings, it may be assumed that 1.0 gr. will Skerman's book and Bergey's Manual. All are displace 1.0 ml. aerobic, and most will grow at room tempera- If counts of any quantitative value are to be ture (some may do better at 370C) on nu- obtained, it is necessary that both the labora- trient agar. tory work and the calculations be done ac- Gram positive rods producing spores: Ba- curately. Since some samples may require high cillus. dilutions, it may be desirable to indicate Gram positive rods without spores: Cory- briefly how these are attained. This procedure nebacterium, Kurthia, Microbacteriumn and is usually accomplished by a series of steps Mycobacterium. with dilutions factors of 1-10 or 1-100 rather Gram negative short rods without spores: than, for example, adding 1 ml. of sample to Achromobacter, Aerobacter, Aeromonas, Ag- 99,999 ml. of diluting fluid to attain a 1- robacterium, Alcaligenes, Chromobacterium, 100,000 final dilution. The latter incorrect Erwinia, Escherichia, Flavobacterium, Proteus, method is not practical for at least two rea- Pseudomonas, Serratia, and Xanthomonas. sons: (1) difficulty of finding container large Gram positive cocci: Aerococcus, Gaffkya, enought to hold 100,000 ml. which can be Micrococcus, Sarcina, Staphylococcus, and sterilized, and (2) difficulty of mixing small Streptococcus. sample and large volume of diluting fluid. The Gram negative cocci: Neisseria. more practical approach might be to use this Gram positive filaments producing chains series of dilutions. of conidia from aerial hyphae: Streptomyces. 1 ml. (or gr.) of sample + 9.0 ml. diluting Quantitative Plating fluid - 10 ml. (1-10 dilution). 1 ml. (1-10) + 9.0 ml. diluting fluid 10 For most purposes at the introductory level, ml. (1-100 dilution). the method of choice for determination of 1 ml. (1-100) 9.0 ml. diluting fluid -10 numbers of bacteria in a given sample is by + ml. (1-1,000 dilution). plating appropriate dilutions of the sample. 1 ml. (1-1,000) + 9.0 ml. diluting fluid This involves the addition of a measured 10 ml. (1-10,000 dilution). amount, usually 1.0 ml., of the dilution of the 1 ml. (1-10,000) + 9.0 ml. diluting fluid sample to the bottom half of a sterile petri 10 ml. (1-100,000 dilution). dish. Approximately 15 ml. of appropriate sterile agar are then poured into the dish, and Thus 5 dilution blanks each containing 9.0 ml. the sample mixed with the agar by gentle ro- of sterile diluting fluid would be needed. A tation of the dish. The total number of different, sterile pipette is, of course, needed colonies will be determined following incuba- for each step. Another procedure for achiev- tion on the assumption that each living cell ing the same dilution but using different vol- will give rise to a colony. Since most samples umes of diluting fluid might be as follows. may contain a large number of cells per unit 1.0 ml. of sample + 99 ml. diluting fluid volume or weight, it is necessary to dilute the - 100 ml. (1-100 dilution). Vol. 22, No. 6 SUGGESTIONS ON BACTERIOLOGICAL TECHNIQUES 351

1.0 ml. ( 1-100) + 99 ml. diluting fluid For this reason, the immediate area, and the 100 ml. (1-10,000 dilution). entire room for that matter, should be kept 1.0 ml. (1-10,000) + 9.0 ml. diluting fluid clear of dust collecting clutter! Before and =10 ml. (1-100,000 dilution). after experiments are done, the table top On some occasions it may not be desirable should be wiped with a cloth (synthetic to use decimal dilutions. The principle illus- sponges deteriorate) moistened with disinfect- trated above may be applied to other numbers, ant. A suitable disinfectant solution may be but be sure to do the calculations correctly as prepared as follows: Add 1 part of mercuric in the following examples of one step in five chloride to 2.5 parts of commercial (technical) different problems. hydrochloric acid. Prepare a small bottle of A. 1 gr. of sample + 14 ml.- 15 ml. (1-15 this stock solution (label: POISON) and for

dilution). use as disinfectant solution, add 2.5 ml. of the Downloaded from http://online.ucpress.edu/abt/article-pdf/22/6/343/16662/4439362.pdf by guest on 29 September 2021 B. 3 gr. of sample + 12 ml. - 15 ml. (1-5 stock solution to one liter of water. Color dilution). with a dye and label POISON. C. 2 ml. (1-100) + 18 ml. - 20 ml. For special work, in order to take extra pre- (1-1,000 dilution). cautions against room air contamination, a D. 1 ml. (1-30) + 7 ml. 8 ml. (1-240 glass or plastic hood is employed. A conven- dilution). ient type may be built in the school shop. This E. 5 ml. (1-50) + 45 ml. 50 ml. (1-500 consists of a rectangular box with sides and dilution). top of glass. The front side should be on If samples of soil, milk, etc., are to be stud- rollers to permit opening so that the arms may ied, the volume of the agar medium in the reach into the work area. An alternate tvpe plate to which the sample is added is not con- utilizes a slanted front-top section which ex- sidered in the calculations. The reason for this tends in the front to about six inches from the is that the number of colonies arising relate to bottom of the hood. This area is left open the number of cells in the aliquot of the final aicross the front to permit access. In such dilution as they are trapped when the agar hoods a microburner replaces the bunsen hardens. On the other hand, if certain con- burner as an aid in avoiding excess heat. centrations of dye, antibiotic, or other chemi- cals are desired in the final medium the References volume of the medium is taken into account. Fagle, D. L. and McClung, L. S.: Bacteriology A Word on LaboratoryCleanliness inoculating devices useful in the biology laboratory. The American Biology Teacher 22:337-338, Nowhere in laboratory work is it more 1960. important to take precautions against dust Paulinus, Sister Mary: Experience with bacteria contamination than in a laboratory where bac- in high school biology. The American Biology teriology experiments are to be performed. Teacher 22:325-332, 1960.

SAB History The Society of American Bacteriologists was founded through the initiative of a volunteer committee consisting of A. C. Abbott, Professor of Hygiene and Bacteriology, University of Pennsylvania; Herbert W. Conn, Professor of Biology, Wesleyan University; and Edwin 0. Jordan, Assistant Professor of Bacteriology, Uni- versity of Chicago. The first meeting was held on December 28, 1899 at New Haven, Connecticut, during the 18th Annual Meeting of the American Society of Naturalists. There were 59 Charter Members. In 1960, the number of Active Mem- bers totaled approximately 6000 and there are 31 Local Branches.