The growth of Microbial Bacteria

1. Each group of learners gets 1 nutrient agar petri dish
2. Using a permanent marker, mark a “T” and the name of the group on the bottom of the dish containing the agar as shown below. (Please note: do these mark on the plastic part of the dish nor in the agar, be careful not to lift the lid as this may contaminate the agar)

3. Dip one sterile swab into a yogurt sample, make sure that not too much yogurt is on the swab as you don’t want it to “blob” on the agar
4. Lifting the lid of the nutrient agar petri dish carefully so that only the sterile swab can fit through (see below), follow the “T” line, do not re-dip your swab into the yogurt.

5. Seal the Petri dish with insulation tape so that air cannot escape or enter the sample. It should ideally be sealed around the whole edge of the Petri dish.
6. Incubate or store in dark room at 20⁰C to 35⁰C for 1 week or until several colonies have developed, note Petri dishes must be stored upside down.
7. Look at sample, and note observations.

Growth 2:

8. Each group gets 2 prepared Rogosa SL agar petri dishes.
9. Looking at the colonies, on that has been grown on the nutrient agar petri dish, look for one that is all by itself, this is the perfect inoculum (see below).

10. Using a sterile swab, carefully swab the inoculum on nutrient agar petri dish.
11. Lifting the lid of the prepared Rogosa SL agar petri dishes carefully so that only the sterile swab can fit through (see below), follow the “T” line.
12. Seal the Petri dish with insulation tape so that air cannot escape or enter the sample. It should ideally be sealed around the whole edge of the Petri dish. Repeat this so that each group has two petri dish from different incolums that has been grown on the nutrient agar petri dish.
13. Incubate or store in dark room at 20⁰C to 35⁰C for 1 week or until several colonies have developed, note petri dishes must be stored upside down.
14. Look both sample and note observations.
15. Prepare a slide from each Rogosa SL agar petri dishes, label each with the groups’ name and sample number on the side of the slide, and place a small drop of water on the slide using graduated pipette.
16. Using a new sterile swab run over a sample scooping up some cells. Rub sample on wet drop on slide and leave to dry in the sun.
17. Put a few drops of bromothyl blue on the slide until sample is blue and rinse sample of excess dye (do so carefully not to remove the smear).
18. Allow the slide to dry in the sun.
19. Place the slide under the microscope and observe the cells take note of the cell shape. The bacteria can be identified under the microscope based on the structure of the microorganism.

Record your observations as follows :

Part 1: Growth of Bacteria in Petri dish with identification labels:

Part 2: Identification of bacteria under the microscope:

CONCLUSION:

Bacteria was grown on 2 different media in order to isolate just the bacteria that originated from the yogurt sample. The samples grown where then dyed and viewed underneath the microscope in order to identify them. The microorganisms should have been identified as lactobacilli bacteria, this means that they would have been rod shaped when viewed underneath the microscope.

PRECAUTIONS:

• When working with bacteria always wear latex gloves
• Do not breathe in any samples
• Do not eat any agar from the petri dish
• Bromothyl Blue solution is hazardous
  • First aid measures
    • Eye Contact: Check for and remove any contact lenses. In case of contact, immediately flush eyes with plenty of water for at least 15 minutes. Cold water may be used. Get medical attention immediately
    • Skin Contact: In case of contact, immediately flush skin with plenty of water for at least 15 minutes while removing contaminated clothing and shoes. Cover the irritated skin with an emollient. Cold water may be used. Wash clothing before reuse. Thoroughly clean shoes before reuse. Get medical attention immediately.
    • Serious Skin Contact: Wash with a disinfectant soap and cover the contaminated skin with an anti-bacterial cream. Seek medical attention.
    • Inhalation: If inhaled, remove to fresh air. If not breathing, give artificial respiration. If breathing is difficult, give oxygen. Get medical attention immediately
    • Serious Inhalation: Evacuate the victim to a safe area as soon as possible. Loosen tight clothing such as a collar, tie, belt or waistband. If breathing is difficult, administer oxygen. If the victim is not breathing, perform mouth-to-mouth resuscitation. Seek medical attention.
    • Ingestion: Do NOT induce vomiting unless directed to do so by medical personnel. Never give anything by mouth to an unconscious person. If large quantities of this material are swallowed, call a physician immediately. Loosen tight clothing such as a collar, tie, belt or waistband.

• Spillage measure
  • Small Spill: Dilute with water and mop up, or absorb with an inert dry material and place in an appropriate waste disposal container. If necessary: Neutralize the residue with a dilute solution of acetic acid.
  • Large Spill: Corrosive liquid. Stop leak if without risk. Absorb with DRY earth, sand or other non-combustible material. Do not get water inside container. Do not touch spilled material. Use water spray curtain to divert vapor drift. Prevent entry into sewers, basements or confined areas; dike if needed. Call for assistance on disposal. Neutralize the residue with a dilute solution of acetic acid. Be careful that the product is not present at a concentration level above TLV. Check TLV on the MSDS and with local authorities