Part IBasic LaboratoryTechniquesand Skills

o isolate, grow, and study microorganisms (specifically bacte-ria, fungi, and protozoa), each species usually is grown in culture.A culture consists of one or more species of microorganisms

growing in a nutrient growth medium usually under specially controlledtemperatures. A culture containing only one species of microorganism iscalled 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 outa few standard procedures (microbiological techniques).

asic Growth Media

Like all living organisms, microbes need nutrients, such as a carbon andnitrogen source as well as vitamins and other growth factors, to survive andgrow. 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 inwhich 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 celldensity. 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 arelatively solid medium on which bacteria and fungi easily grow. Agar is apolymer of galactose that is extracted from the cell walls of seaweed (redalgae); agar has no nutritional value in an agar culture. In solution, powderedagar liquefies at 100°C and then will solidify at 40°C as the solution cools. Thismeans microorganisms isolated from humans can be cultivated at humanbody temperature (37°C) without fear of the medium liquefying.

B

T

4 1

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

Different types of agar media can be prepared (Figure I.2). While in theliquefied state, the medium is sterilized; that is, all living organisms andviruses are killed and no spores exist. If such a medium is placed in testtubes and allowed to cool and harden in a slanted position, agar slants areproduced. These are useful for maintaining pure cultures. Similar tubesallowed to harden in the upright position are called agar deeps. If liquidagar 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 individualcolonies; that is, clusters of cells derived through multiplication from a singlecell or spore. For example, a soil sample placed in a sterile broth medium would

2 4

primarily used for

MICROBIOLOGICAL CULTURE MEDIA

Solution of growth nutrients

Liquid media

Agar platesAgar deepsAgar slantsBroths

Solid media

consist of

with agarwithout agar

in the form of

primarily used for

can be in the form of

Maintaining purecultures

Maintaining purecultures;

physiological studies

Physiological studies

Isolation of pure cultures and study of

microbial diversity

F I G U R E I . 1A Concept Map for Microbiological Media. The different types of microbiologicalmedia and their uses are shown.

Side view Front view

(a) Agar slants (b) Agar deep tube (c) Agar plate

F I G U R E I . 2The 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 2

produce a dense population of cells. The same sample on an agar medium willshow the diversity of bacteria and fungi in the sample.

• Different species often produce unique colony characteristics that can aid inidentification. Therefore, most bacterial species can be visually separated fromfungi, and even different species of bacteria can be told apart by the way thecolonies appear (Exercise 2).

• Since the colony represents a single species, a sample from a well-isolated colonycan be transferred to a sterile broth or agar medium to form a pure culture.

ulture Equipment

During your work in the microbiology laboratory, cultures will be studiedor maintained in different types of culture equipment.

• Culture tubes. Broth cultures typically use glass tubes with a suitable sleeve-likecap to keep out contaminating microbes. Built-in ridges on tube closures allowentry of air. Some broth media may use screw-cap tubes. Agar slants and agardeeps 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 aslightly larger, loose-fitting lid covers the plate and again keeps out contaminants.Once inoculated with a microorganism, the dishes usually are incubated in aninverted position (lid down) to prevent condensation forming on the cover. Leftright-side up, the condensation could drop onto the agar surface and makeculture studies difficult.

ransfer Instruments

Microorganisms must be transferred from one vessel to another or fromstock cultures to various media for maintenance, growth, and study (Exer-cise 1). Such a transfer is called subculturing and must be carried outunder aseptic conditions; that is, transfer between nutrient media with-out contamination with other microorganisms. The most commonly usedinstruments 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 organismis aseptically placed on the loop and streaked or suspended in an appropriateagar or broth medium; needles with organism usually are stabbed into an agardeep. Disposable loops or needles also may be used.

• Pipettes can be used for sterile transfers (Figure I.3c, d). Sterile plastic or glasspipettes draw up liquids into the graduated (calibrated) column. The graduationsmark the different volumes that can be delivered. Your instructor will demon-strate the proper use for the pipettes.

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 optimumtemperature necessary for growth. Since most incubators use dry heat, which

G

T

C

4 3

!Never pipette chemical solutions or broths bymouth! All pipetting should be done with the aid of mechanical pipettedevices.

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

can dehydrate the agar or broth medium, a beaker of water can be placed inthe 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 onlyfor 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 after24 to 48 hours at 37°C, the cultures will be moved to a refrigerator, whichwill stop or greatly slow down microbial growth.

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.

L

4 4

Loop

Needle

Shaft

Handle

(a) Inoculation needle

(b) Inoculation loop

(c) Blow-out 1 ml pipette

(d) To-deliver 10 ml pipette

.9

9

10

.8

8

.7

.6

.5

7

6

5

.44

.3 3

.2 2

.1 1

0

TD

1 IN

1/1

00 m

l

TD20 C10 ml

10 IN

1/1

0 m

l

Etched ringon mouthpiece(blow out)

0.01 ml eachminor division

0.1 ml eachminor division

0.1 ml majordivisions

Identificationand graduations

No etched ringon mouthpiece(to deliver)

1 ml majordivisions

Final few dropsmust be blownout to deliverindicated volume

F I G U R E I . 3Microbiological Transfer Instruments. Inoculating loops and needles, and pipettes,are among the more common items used.

43038_CH01_0001.qxd 1/3/07 3:41 PM Page 4

CultureTransferTechniques

n several microbiology exercises and experiments, you will needto transfer microorganisms from one medium to another bysubculturing. This aseptic technique also is commonly used

when preparing and maintaining stock cultures, and when carrying out anumber 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 transferredusing proper aseptic techniques.

Aseptic transfer techniques are not difficult to learn and perform, althoughsome eye-hand coordination and practice may be needed. To simplify theprocess:

• Make sure all needed materials, cultures, and media are ready, and the wire ofthe transferring device (inoculating loop or needle) is straight.

• Label appropriately all media into which microbes will be transferred so you canidentify your cultures during the lab period or, if incubated, when the next labperiod meets.

Aseptic Transfer Techniques

The three steps of the aseptic transfer process are described below andshown in Figure 1.1 for transferring bacteria. The description and figureshow the use of the inoculating loop, which is typically used for broth andagar slant/plate transfers. Read through the process completely beforeattempting the procedures described below.

1. Holding the tubes and sterilizing the inoculating loop

• If you are right-handed, hold the stock culture tube (from which you willmake the transfer) and the tube to be inoculated in the palm of your lefthand (Figure 1.1a). (If you are left-handed, the tubes should be in yourright hand).

• The inoculating loop is not sterile. To sterilize it, hold the handle of theinstrument in your free hand like you would a pen or pencil. Place the loop

A.

I

1C U L T U R E T R A N S F E R T E C H N I Q U E S 1 5

PURPOSE: to perform asep-tic transfers of bacteria frombroth to agar slant and fromagar slant to broth.

43038_CH01_0001.qxd 1/3/07 3:41 PM Page 5

in the hottest portion of the Bunsen burner flame, which is the top of theblue flame (Figure 1.1b). (If you are using incinerators, your instructor willdescribe the sterilization process.) In a few seconds the entire loop will turnred 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 holdit in your hand and allow the loop or needle to cool for 10 to 20 seconds.

2. The transfer processYou are now ready to carry out the transfer. Two examples are describedand illustrated: one transfer is from a broth to an agar slant and the otheris from an agar slant to a broth. Inoculation of an agar plate is described inExercise 2.

• The caps or closures of the two tubes are removed by grasping the cap of theleft tube between your little finger and ring finger; the cap of the right tubebetween the ring finger and middle finger. Now remove the caps and brieflyflame 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 zigzagmotion 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 surfacewhere the bacteria are growing. Remove just a minute sample of bacteria(Figure 1.1e2).

• The loop is immediately inserted into the broth tube. Shake the loop gentlyto dislodge the bacteria and suspend them in the broth. Withdraw the loopfrom the broth tube.

3. Capping tubes and re-sterilizing the loop

• Following the transfer, the necks of the tubes are briefly reflamed and thecaps 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 theloop (Figure 1.1h).

• Place the tubes in the test tube rack or appropriate tube holder.

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

rocedure

1. Label all tubes of sterile media as described above (page 5).

2. Following the procedure outlined and illustrated, perform the followingaseptic 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.

P

S

6 1 C U L T U R E T R A N S F E R T E C H N I Q U E S

!Do not fill the entire loopwith bacteria.

43038_CH01_0001.qxd 1/3/07 3:41 PM Page 6

C U L T U R E T R A N S F E R T E C H N I Q U E S 1 7

a b

Step

1

cd

e2

2

3

f

e1

h

g

F I G U R E 1 . 1(a) Hold the tubes in your hand, using your thumb to separate the tubes. (b) Sterilize the loop by incineration in the innerblue (hottest) part of the flame. (c) Remove the caps from the tubes. (d) Briefly pass the tue necks through the Bunsenburner flame. (e1) Transfer a loopful of broth from a broth tube to an agar slant. (e2) Transfer a small inoculum of bacteriafrom an agar slant to a broth tube. Gently mix. (f) Briefly pass the tube necks through the Bunsenburner 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.

3. Incubate all cultures at 25°C for 24 to 48 hours. (If lab does not meet untilthe following week, arrangements should be made to move the cultures toa refrigerator after the incubation period.)

4. In the next lab period:

• Examine the broth culture for the appearance of growth. Growth is indi-cated by the broth appearing fairly turbid; that is, the liquid should bedense and cloudy.

• Examine the agar slant culture for the appearance of growth. Growth onagar can be seen by noting the appearance of a thick, red film of bacteria onthe surface of the slant.

• Record your observations in the Table 1.1

Quick ProcedureAseptic TransferTechnique

1. Sterilize inoculatingloop.

2. Transfer broth film orsmall bacterial sampleon loop from stock tube(broth or agar slant) oragar plate to appropri-ate sterile tube (mix inbroth; streak on agarslant or plate).

3. Resterilize loop.

43038_CH01_0001.qxd 1/3/07 3:41 PM Page 7

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 certaintythata. 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.

Q

8 1 C U L T U R E T R A N S F E R T E C H N I Q U E S

43038_CH01_0001.qxd 1/3/07 3:41 PM Page 8

Name

Date Section

Exercise Results

Culture Transfer Techniques

A. Aseptic Transfer Techniques

1

Medium Subcultured

NutrientBroth

Nutrient AgarSlant

Growth(+) or (–)

Draw thedistribution ofgrowth.

Describe theappearanceof bacterialgrowth

C U L T U R E T R A N S F E R T E C H N I Q U E S 1 9

43038_CH01_0001.qxd 1/3/07 3:41 PM Page 9

43038_CH01_0001.qxd 1/3/07 3:41 PM Page 10