Practical 4 Aseptic techniques and Inoculation techniques

Objectives:
By the end of this practical session, you will be able to: Practice the use of aseptic techniques in microbiology. Perform inoculation by plating technique. Perform inoculation of slopes and stabs. Perform inoculation of broths.

Introduction:
When working in microbiology laboratory, aseptic techniques must be practiced to prevent contamination of cultures and specimens, and to prevent the worker and the environment from infection.

Aseptic techniques:
Flame sterilize wire loops, straight wires, forcepsetc, before and after use. Flame the neck of specimen bottles, culture bottles, and tubes after removing and before replacing the caps, bungs and plugs. Avoid touching the caps of tubes and bottles to touch an unsterile surface when inoculating. Always use racks to hold tubes and bottles. Make slide preparations from specimens after inoculating the culture medium. Decontaminate workbench before starting work and after finishing. When working with hazardous pathogens, use a safety cabinet. Wear protective clothing, wash hands Never mouth pipette, eat, drink or smoke in the laboratory.

Inoculation:
Several methods are used to inoculate the organisms to the culture media. They are: Inoculation of media in Petri dishes: Lawn/carpet culture Stroke culture Stab culture Pour plate culture: Liquid (broth) cultures

Incubation:
Inoculated media should be incubated as soon as possible, at a suitable temperature, humidity and gaseous atmosphere. The length of incubation depends on the time the bacteria take to develop the colonies. Optimum temperature: the temperature at which an organism grows best. The selected temperature range for most routine culturing is 35-37C, with 36C being recommended by most microbiologists. Temperature of growth is also used in differentiating some organisms. The atmosphere should not be too dry. It can affect the growth and viability of many organisms. Microorganisms vary in their need for oxygen. The most suitable environment (aerobic or anaerobic..etc) should be provided for the growth of the organisms.

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Pre-lab exercise:
Using a dictionary or a microbiology text book find the meanings of the following terms: Aseptic techniques

Inoculation

Turbid

CLED media

Viable

Exercise 1: Aseptic techniques

Aim:

Materials:
3 sterile Bijoux bottle 20 ml sterile nutrient broth Viable and killed broth cultures of E.coli Sterile Disposable Pasteur pipettes

Method:
1. Using aseptic techniques as demonstrated, transfer 2ml of sterile nutrient broth into each sterile bijoux bottle. 2. Label the bottles (not the caps!), with a marker pen as 1, 2 & 3. 3. Inoculate the bottles by placing a drop of killed E.coli in bottle 1 and live culture in bottle 2 using sterile Pasteur pipettes. 4. Leave bottle 3 without inoculating, as a negative control. 5. Incubate all bottles at 35C for 3 days. 6. in the next practical, examine the cultures for growth and record the results as: Turbid (growth) = positive Clear (no growth) = negative If glucose bromothymol blue is used instead of nutrient broth, a color change in the broth from blue-green to yellow indicates that glucose has been fermented as a result of bacterial growth. The acid produced altered the pH of the broth and caused the color to change.

Results:
Killed E.coli Bottle Growth 1 Live E.coli 2 Control 3

Conclusion:
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Comment on your aseptic techniques.

Exercise 2: Plating techniques

Materials:
Agar plates Inoculating loops 2 Swab specimens & urine specimen Bunsen burner

Method:
1. Label the base of the blood agar plate with your name and specimen number. 2. Using a marker pen, divide the second blood agar plate into two halves and label each half with the specimen number and your name. 3. Flame sterilize the inoculating loop and let it cool. 4. Place a small amount of inoculum near the periphery of plate 1, with a loop or swab. 5. Complete streaking by the whole plate method (appendix 3). 6. Using the half- plate method, inoculate on both halves of plate 2. 7. Inoculate the CLED plate by streaking as shown in figure 1. 8. Incubate the plates at the appropriate temperature and atmosphere, for the desired period of time.

Figure 1

Exercise 3: Inoculation of stabs and slopes

Materials:
Agar slants and butts Inoculating loops and straight wires Broth culture of E.coli Bunsen burner

Method:
1. 2. 3. 4. 5. Flame sterilize the inoculating wire. Pick up the inoculum with the sterilized loop. Remove the cotton plug or cap with the little finger of the hand with which the loop is held. Flame the neck of the tubes or bottles, by passing it through the flame 2-3 times. Transfer the inoculum to the medium, keeping the tubes in a nearly horizontal position as follows: Slopes: first make a straight line of inoculation over the surface of the media and then make the streaking over the straight line with a ziz-zag motion. Butts: using a straight inoculating wire, stab deep into the butt through the centre.
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6. 7. 8. 9. 10.

Re-flame the neck of the bottles/tubes. Re-cap or plug the tubes/bottles. Flame the inoculating wire and replace it. Replace the tubes in a test tube rack. Incubate the media at the appropriate temperature and atmosphere, for the desired period of time.

Exercise 4: Inoculation of broths

Materials:
2 nutrient broths Inoculating loops 2 pre-inoculated culture plates Bunsen burner

Method:
1. 2. 3. 4. 5. 6. 7. 8. 9. Label the culture bottle. Flame sterilize the inoculating wire. Allow it to cool. Remove the cotton plug or cap. Flame the neck of the tubes or bottles. Pick 2-3 colonies from the pure culture plate. Transfer the inoculum into the broth. Re-flame the neck of the bottles/tubes. Re-cap or plug the tubes/bottles. Incubate the media at the appropriate temperature and atmosphere, for the desired period of time.

Conclusion:

Questions:
1. Describe the proper method for flame sterilizing an inoculating loop. 2. Why should the loop be flame sterilized in between streaks while streaking on agar plates? 3. State any 3 aseptic techniques that would help prevent contamination in the microbiology laboratory. 4. What are aerosols? And how can aerosols be created in microbiology laboratory? 5. How would you prepare a pour plate and spread plate culture? 6. At what temperature and for how long are the cultures incubated routinely?

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