Part I Basic Laboratory Techniques and Skills

43038_CH01_0001.qxd 1/3/07 3:41 PM Page 1

4 1 Part I Basic Laboratory Techniques and Skills

o isolate, grow, and study microorganisms (specifically bacte- ria, fungi, and protozoa), each species usually is grown in culture. T A culture consists of one or more species of microorganisms growing in a nutrient growth medium usually under specially controlled temperatures. A culture containing only one species of microorganism is called a pure culture. To isolate and grow microorganisms in pure culture, you will need a variety of materials and equipment and the skills to carry out a few standard procedures (microbiological techniques).

B asic Growth Media Like all living organisms, microbes need nutrients, such as a carbon and nitrogen source as well as vitamins and other growth factors, to survive and grow. The source for these nutrients usually comes from the enzymatic degra- dation of other complex nutrients derived from plant and animal sources. The composition of the nutrients represents a culture medium (Figure I.1). Such media may be in the form of a liquid or solid. A liquid solution in which microbes, especially bacteria and protozoa, will grow is called a nutri- ent broth. A broth medium is one way to grow microorganisms to a high cell density. However, if the broth contains more than one species of microor- ganism, each individual species cannot be differentiated with the naked eye. A broth supplemented with a solidifying agent, called agar, produces a relatively solid medium on which bacteria and fungi easily grow. Agar is a polymer of galactose that is extracted from the cell walls of seaweed (red algae); agar has no nutritional value in an agar culture. In solution, powdered agar liquefies at 100°C and then will solidify at 40°C as the solution cools. This means microorganisms isolated from humans can be cultivated at human body temperature (37°C) without fear of the medium liquefying. 43038_CH01_0001.qxd 1/3/07 3:41 PM Page 2

2 4

MICROBIOLOGICAL CULTURE MEDIA consist of Solution of growth nutrients

without agar with agar

Liquid media Solid media

in the form of can be in the form of

Broths Agar slants Agar deeps Agar plates

primarily used for primarily used for

Maintaining pure Maintaining pure Physiological Isolation of pure cultures cultures; studies cultures and study of physiological studies microbial diversity

FIGURE I.1 A Concept Map for Microbiological Media. The different types of microbiological media and their uses are shown.

Different types of agar media can be prepared (Figure I.2). While in the liquefied state, the medium is sterilized; that is, all living organisms and viruses are killed and no spores exist. If such a medium is placed in test tubes and allowed to cool and harden in a slanted position, agar slants are produced. These are useful for maintaining pure cultures. Similar tubes allowed to harden in the upright position are called agar deeps. If liquid agar medium is poured into culture (Petri) dishes and allowed to harden, agar plates are formed. Agar plates provide a large surface area for the iso- lation and study of microorganisms. The agar medium has many advantages: • It represents a hardened surface on which bacteria or fungi can grow as individual colonies; that is, clusters of cells derived through multiplication from a single cell or spore. For example, a soil sample placed in a sterile broth medium would

Side view Front view

(a) Agar slants (b) Agar deep tube (c) Agar plate FIGURE I.2 The Different Types of Solid Media. Solid media can be prepared using agar to pre- pare (a) agar slants, (b) agar deeps, and (c) agar plates. 43038_CH01_0001.qxd 1/3/07 3:41 PM Page 3

4 3

produce a dense population of cells. The same sample on an agar medium will show the diversity of bacteria and fungi in the sample. • Different species often produce unique colony characteristics that can aid in identification. Therefore, most bacterial species can be visually separated from fungi, and even different species of bacteria can be told apart by the way the colonies appear (Exercise 2). • Since the colony represents a single species, a sample from a well-isolated colony can be transferred to a sterile broth or agar medium to form a pure culture.

C ulture Equipment During your work in the microbiology laboratory, cultures will be studied or maintained in different types of culture equipment. • Culture tubes. Broth cultures typically use glass tubes with a suitable sleeve-like cap to keep out contaminating microbes. Built-in ridges on tube closures allow entry of air. Some broth media may use screw-cap tubes. Agar slants and agar deeps also use culture tubes either with a sleeve-like cap or a screw cap. • Culture plates. Plastic culture (Petri) dishes are used to cultivate micro- organisms. The solidified medium is in the bottom half of the plate, while a slightly larger, loose-fitting lid covers the plate and again keeps out contaminants. Once inoculated with a microorganism, the dishes usually are incubated in an inverted position (lid down) to prevent condensation forming on the cover. Left right-side up, the condensation could drop onto the agar surface and make culture studies difficult.

T ransfer Instruments Microorganisms must be transferred from one vessel to another or from stock cultures to various media for maintenance, growth, and study (Exer- cise 1). Such a transfer is called subculturing and must be carried out under aseptic conditions; that is, transfer between nutrient media with- out contamination with other microorganisms. The most commonly used instruments are the following: • Wire inoculating loops and needles are used to perform transfers (Figure I.3a, b). They are easily sterilized by incineration in the blue (hottest) portion of the Bunsen burner flame. After slight cooling, a sample of organism is aseptically placed on the loop and streaked or suspended in an appropriate agar or broth medium; needles with organism usually are stabbed into an agar deep. Disposable loops or needles also may be used. • Pipettes can be used for sterile transfers (Figure I.3c, d). Sterile plastic or glass Never pipette chemical pipettes draw up liquids into the graduated (calibrated) column. The graduations solutions or broths by mouth! All pipetting mark the different volumes that can be delivered. Your instructor will demon- should be done with the strate the proper use for the pipettes. aid of mechanical pipette devices. G rowth and Incubation Appliances ! Many microorganisms have specific temperature requirements for growth. In the entry-level course, most organisms can be grown at either room tem- perature (20–22°C) or human body temperature (37°C). Maintaining the lat- ter temperature for long periods of time, such as between lab sessions, requires putting the cultures in an incubator that maintains the optimum temperature necessary for growth. Since most incubators use dry heat, which 43038_CH01_0001.qxd 1/3/07 3:41 PM Page 4

4 4

Loop No etched ring Etched ring on mouthpiece Needle on mouthpiece (to deliver) (blow out)

Identification 10 IN 1/10 ml TD and graduations 20 C 10 ml TD 1 IN 1/100 ml 0

.1 1 0.1 ml major .2 divisions 2 1 ml major 0.01 ml each divisions Shaft minor division .3 3

4 .4 0.1 ml each minor division 5 .5

6 .6

7 .7 Handle 8

.8 9

Final few drops .9 must be blown 10 out to deliver indicated volume

(a) Inoculation (b) Inoculation (c) Blow-out (d) To-deliver needle loop 1 ml pipette 10 ml pipette FIGURE I.3 Microbiological Transfer Instruments. Inoculating loops and needles, and pipettes, are among the more common items used.

can dehydrate the agar or broth medium, a beaker of water can be placed in the incubator during the growth period to maintain a moist environment. For short periods of time, such as during a lab exercise or experiment, cultures may be incubated in a waterbath, where the appropriate tem- perature is maintained by a heating device. A waterbath can be used only for cultivation of organisms in a broth medium. Since your lab session might meet only once a week, cultures incu- bated at 37°C for seven days might overgrow the tube or plate. Often after 24 to 48 hours at 37°C, the cultures will be moved to a refrigerator, which will stop or greatly slow down microbial growth.

L earning Objectives When you have completed the exercise in Part I, you should be capable of • Completing the aseptic transfer of microorganisms. • Carrying out the pure culturing procedures to separate bacteria in a mixed population. 43038_CH01_0001.qxd 1/3/07 3:41 PM Page 5

CULTURE TRANSFER TECHNIQUES 1 5 Culture 1 Transfer Techniques

n several microbiology exercises and experiments, you will need to transfer microorganisms from one medium to another by I subculturing. This aseptic technique also is commonly used when preparing and maintaining stock cultures, and when carrying out a number of microbiological test procedures. Microorganisms are everywhere, including in the air of your course lab, as well as on the floors, bench tops, and equipment. If appropriate precau- tions are not taken, these microbes could end up in one of your subcultures; that is, microbes could contaminate your materials. To prevent contam- ination by unwanted microbes, the microbes you want must be transferred using proper aseptic techniques. Aseptic transfer techniques are not difficult to learn and perform, although some eye-hand coordination and practice may be needed. To simplify the process: • Make sure all needed materials, cultures, and media are ready, and the wire of the transferring device (inoculating loop or needle) is straight. • Label appropriately all media into which microbes will be transferred so you can identify your cultures during the lab period or, if incubated, when the next lab period meets.

A. Aseptic Transfer Techniques

The three steps of the aseptic transfer process are described below and shown in Figure 1.1 for transferring bacteria. The description and figure show the use of the inoculating loop, which is typically used for broth and agar slant/plate transfers. Read through the process completely before attempting the procedures described below.

1. Holding the tubes and sterilizing the inoculating loop PURPOSE: to perform asep- If you are right-handed, hold the stock culture tube (from which you will tic transfers of bacteria from • broth to agar slant and from make the transfer) and the tube to be inoculated in the palm of your left agar slant to broth. hand (Figure 1.1a). (If you are left-handed, the tubes should be in your right hand). • The inoculating loop is not sterile. To sterilize it, hold the handle of the instrument in your free hand like you would a pen or pencil. Place the loop 43038_CH01_0001.qxd 1/3/07 3:41 PM Page 6

6 1 CULTURE TRANSFER TECHNIQUES

in the hottest portion of the Bunsen burner flame, which is the top of the blue flame (Figure 1.1b). (If you are using incinerators, your instructor will describe the sterilization process.) In a few seconds the entire loop will turn red hot. When it does, move the rest of the wire rapidly through the flame. • Once sterilized, the loop should not be placed on the lab bench. Simply hold it in your hand and allow the loop or needle to cool for 10 to 20 seconds. 2. The transfer process You are now ready to carry out the transfer. Two examples are described and illustrated: one transfer is from a broth to an agar slant and the other is from an agar slant to a broth. Inoculation of an agar plate is described in Exercise 2. • The caps or closures of the two tubes are removed by grasping the cap of the left tube between your little finger and ring finger; the cap of the right tube between the ring finger and middle finger. Now remove the caps and briefly flame the necks of the tubes in the Bunsen burner flame (Figure 1.1c, d). Broth to agar slant • Insert the loop into the broth tube and withdraw a loopful of liquid (Figure 1.1e1). You should see a watery film on the loop. • The broth-containing loop is immediately inserted into the agar slant tube. Place the loop flat on the agar surface and move the loop rapidly in a zigzag motion gently across the agar surface from bottom to top. Remove the loop. Agar slant to broth • Insert the loop into the agar slant and touch the loop to the agar surface where the bacteria are growing. Remove just a minute sample of bacteria (Figure 1.1e2). Do not fill the entire loop • The loop is immediately inserted into the broth tube. Shake the loop gently with bacteria. to dislodge the bacteria and suspend them in the broth. Withdraw the loop from the broth tube. 3. Capping tubes and re-sterilizing the loop • Following the transfer, the necks of the tubes are briefly reflamed and the ! caps placed back on the respective tubes from which they were removed (Figure 1.1f, g). • The loop is again flamed to incinerate any remaining bacteria still on the loop (Figure 1.1h). • Place the tubes in the test tube rack or appropriate tube holder.

S pecial Materials • Nutrient broth and nutrient agar slant cultures • One 24-hour nutrient broth of Serratia marcescens (or other bacterial species) per student • One 24-hour nutrient agar slant of Serratia marcescens (or other bacterial species) per student

P rocedure 1. Label all tubes of sterile media as described above (page 5). 2. Following the procedure outlined and illustrated, perform the following aseptic transfers: a. Transfer S. marcescens from a broth culture to a nutrient agar slant. b. Transfer S. marcescens from an agar slant culture to a nutrient broth. 43038_CH01_0001.qxd 1/3/07 3:41 PM Page 7

CULTURE TRANSFER TECHNIQUES 1 7

Step

1

a b

c d

2

e1 e2

3

h f g FIGURE 1.1 (a) Hold the tubes in your hand, using your thumb to separate the tubes. (b) Sterilize the loop by incineration in the inner blue (hottest) part of the flame. (c) Remove the caps from the tubes. (d) Briefly pass the tue necks through the Bunsen burner flame. (e1) Transfer a loopful of broth from a broth tube to an agar slant. (e2) Transfer a small inoculum of bacteria from an agar slant to a broth tube. Gently mix. (f) Briefly pass the tube necks through the Bunsen burner flame. (g) Replace the caps on the appropriate tubes. (h) Resterilize the loop by incinera- tion in the inner blue (hottest) part of the flame. Quick Procedure Aseptic Transfer Technique 3. Incubate all cultures at 25°C for 24 to 48 hours. (If lab does not meet until 1. Sterilize inoculating the following week, arrangements should be made to move the cultures to loop. a refrigerator after the incubation period.) 2. Transfer broth film or small bacterial sample 4. In the next lab period: on loop from stock tube • Examine the broth culture for the appearance of growth. Growth is indi- (broth or agar slant) or cated by the broth appearing fairly turbid; that is, the liquid should be agar plate to appropri- dense and cloudy. ate sterile tube (mix in • Examine the agar slant culture for the appearance of growth. Growth on broth; streak on agar agar can be seen by noting the appearance of a thick, red film of bacteria on slant or plate). the surface of the slant. 3. Resterilize loop. • Record your observations in the Table 1.1 43038_CH01_0001.qxd 1/3/07 3:41 PM Page 8

8 1 CULTURE TRANSFER TECHNIQUES

Q uestions 1. What is meant by an aseptic transfer? 2. Explain why it is necessary to: a. Flame the inoculating loop before and after each inoculation. b. Cool the inoculating loop prior to obtaining the bacterial sample. 3. Why was S. marcescens used in this exercise? 4. From your answer to question 3, can you say with a high degree of certainty that a. The broth culture is a pure culture of S. marcescens? b. The agar slant is a pure culture of S. marcescens? 5. Which transfer process was more difficult to perform? Explain. 43038_CH01_0001.qxd 1/3/07 3:41 PM Page 9

CULTURE TRANSFER TECHNIQUES 1 9

Name

Date Section

Exercise 1 Results Culture Transfer Techniques

A. Aseptic Transfer Techniques

Medium Subcultured

Nutrient Nutrient Agar Broth Slant

Growth (+) or (–)

Draw the distribution of growth.

Describe the appearance of bacterial growth 43038_CH01_0001.qxd 1/3/07 3:41 PM Page 10