Microbiology PR5 Antibiotic Detection Methods PDF

Microbiology PR5 ================ (i). Assay for the production of antibiotics by soil fungi using an =================================================================== **agar overlay technique.** This technique allows the production of a lawn of bacteria within a thin layer of agar across the...

Microbiology PR5 ================ (i). Assay for the production of antibiotics by soil fungi using an =================================================================== **agar overlay technique.** This technique allows the production of a lawn of bacteria within a thin layer of agar across the surface of a plate. Bacteria are added to a soft top agar (0.75% agar, as opposed to the usual 1.5% for agar plates) which has been melted at 100°C and cooled to 45°C. This is warm enough so the agar remains liquid, but cool enough so that the bacteria are not killed (for a period of time). The melted agar/bacterial suspension is mixed and poured evenly across the top of an agar plate and allowed to solidify. The bacteria distributed through the top agar will grow to produce a turbid lawn. If the freshly seeded lawn is exposed to various antibacterial agents produced by fungal or bacterial colonies underneath this layer while being incubated at 37°C, any inhibition of bacterial growth in the top layer will cause a reduction in the turbidity of the lawn above the colony. (i). Assay for the production of antibiotics by soil fungi using an agar overlay ================================================================================ technique. ========== You are given 10mls of soft agar (1.0%) held at 45^o^C in a waterbath. You are given an overnight bacterial culture of *Micrococcus luteus* and *E. coli*. Prepare 1% bacterial cultures (indicator culture) of **EACH** in 10ml soft agar. What quantity of overnight culture is added to the 10ml of soft agar? Show your calculations. Method: ======= 1. Examine the soil plates from last week's practical. =================================================== 2. Choose plates that has well isolated colonies and a variety of morphological types. Choose two plates. One will be overlaid with *M. luteus* and the other overlaid with a culture of *E. coli*. ================================================================================================================================================================================================ http://www.rothamsted.bbsrc.ac.uk/ppi/rhizo2/images/Picture5.jpg  **Cultured soil bacteria** 3. Overlay these plates with approximately10ml of the prepared inoculum of *M. luteus* and *E. coli*.  4. Allow the plates to stand on the bench for 30 minutes to dry. 5. Incubate the plates at 37^o^C (temperature suitable for the indicator strain) for 18h, remove from the incubator and place at 4^o^C until examined. 6. Observe zones of inhibition (clear circles in a lawn of bacterial growth). Methods for obtaining well isolated single colonies. ==================================================== Streaking methods using a wire inoculation loop. ================================================ Introduction: In order to ensure that a culture is pure it must be grown from a single cell. As many billions of cells are present in as little as 1cm^3^ of an overnight culture of micro-organisms, special streaking techniques are used to isolate a single cell. The number of cells present in the culture or sample being used will have a bearing on the choice of method. Materials: 1 Petri-dishes of Nutrient agar 2 Wire loop 3 Overnight culture of *E. coli* 4 **Sour milk, pasteurised milk, *E.coli.*** Method: **Where the sample contains \< 50,000 cfu.cm**3 1 Sterilise the loop and aseptically inoculate with pond water sample. 2 Streak the petri-dish in a zigzag manner as shown, using the full width of the plate without recrossing. **Where the sample contains\> 50,000 cfu.cm3** 1 Sterilise the loop and inoculate with the overnight culture provided. 2 Perform a first streak (A) as shown. Then re sterilise the loop 3 **Without re inoculating the loop**, streak across the plate through the first streak once then continue in a zigzag manner as before. (B). 4 Repeat step 3, passing through the second streak and zigzagging as before. (C) An alternative method is as follows: 1 Sterilise and inoculate the loop withthe overnight culture provided. Streak the loop across the surface of the plate in parallel lines (X). Sterilise the loop. 2 Without re inoculating the loop, streak across the plate in parallel lines at an angle to the first set of lines and crossing them (Y). Sterilise the loop. 3 Repeat step 2 a third, and if possible, a fourth time (Z). Incubate all plates at 37oC for 24 hrs. Pasteurised Milksample : Single streak technique: Sour Milk Sample: \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ *E. coli*Sample: \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ **QUADRANT Streak Method.** Results: Time of incubating: \_\_\_\_\_\_\_\_\_\_\_\_\_ Date: \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ Time of reading: \_\_\_\_\_\_\_\_\_\_\_\_\_ Date: \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ Total incubation time: \_\_\_\_\_\_\_ Temp: \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ Pasteurised Milk sample : **Quadrant streak technique**: Sour Milk Sample: \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ *E. coli* Sample: \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ Discussion: (Include a discussion which method gave the best results and why.) Conclusion: \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ **Questions:** Give reasons why we often wish to obtain isolated colonies in microbiology. \_ How would you treat a solid food sample, eg.sausagemeat, so that you might obtain isolated colonies from the sample ?