Question

Question 1:
Why a pure colony isolation on the plate is necessary?
Question 2:
Briefly define the use of the following media in combination with primary culture media for
isolation of organisms from clinical specimens. Support your answer by quoting examples for
each category: (1.5 x 4 = 6 Marks)
1. An enrichment broth
2. Nutritional agar
3. Selective agar
4. Differential media
Question 3:
What are the limitations associated with placing too much organism on the slide prior to staining
and placing too little organism on the slide prior to staining?
Question 4:
Define the term antibody titer. What is significance of antibody titer determination in "paired
sera" while addressing the etiology of an infectious disease?
Question 5:
While performing antimicrobial sensitivity testing by Kirby Baeur Disc diffusion method, If you
mistakenly adjusted the organism inoculum suspension to be equal to a number 1 McFarland
turbidity standard, would the results be falsely resistant, sensitive, or not altered. Give reason.
(1.5 marks)
Question 6:
Small colonies are observed at the inner edge of the zone of inhibition around the oxacillin disk
when S. aureus is tested. The outer zone size corresponds to an interpretation of susceptible, and
the small colonies are growing in the resistant range. How will you interpret the results—S
(sensitive), I (intermediate), or R (resistant)? Give reason. (1.5 Marks)

Answer 1

1) It is extremely necessary to obtain a pure colony isolation on the agar plates. This will not only help in the identification and detection of the micro-organisms from a sample or specimen of mixed cultures but also help in the quantification of the isolates. This is crucial for performing a series of biochemical tests and also the genetic tests which require no contamination whatsoever. We would not want false positives or any kind of wrong reult hence it is impotant that a pure colony isolation is carried out.

2) The follwing are the examples of the various media used and their uses respectively:

1) Enrichment broth is a liquid media which facilitates the growth of the micro-organisms due to the nutrients which are present to enrich the growth of the microbes. The media is enriched by the addition of ingredients like meat, vegetable matter etc. An example is Mueller Hinton broth.

2) The nutritional agar example is ntrient agar which has robust ingredients like peptone and beef extract. It allows the growth of all kinds of micro-organisms and is thus used for a general purpose.

3) Selective agar is a form of growth medium which selects a particular group of micr-organisms and does not allow the growth of the other organisms. The example is a Tellurite agar which is selective for the growth of gram-postive organisms.

4) Differential growth medium helps in the growth of micro-organisms by differentiating between notable parameters of their growth patterns and infection rates. An example is blood agar where sheep blood can be used and helps in differentiating between types of hemolysis for all the organisms which are able to grow in this nutrient-rich enviornment.

3) It is necessary to understand that while performing a gram staining procedure, the slide should be prepared with appropriate and optimum amount of culture since too little and too much of the sample can only hamper the outcome being visualised under the microscope. the individual bacterial cells will not be stained equally throughout and it will even be difficult to fix the cover slip over the specimen placed on the slide. Since the specimen cannot be fixed it will affect the end result. Further if the bacterial cells are not fixed to the slide then they will get washed off during the application of the gram stain step so it is necessary that only small amounts of the sample using the nichrome loop is taken on the slide.

4) The antibody titer is the value which indicates the number of antibodies present in the blood sample. The antibodies are produced in response to the antigens which are presnt and have been introduced into the body. the titer values are very crucial in the case of paired sera since they are mainly used as a diagnostic test and helps in determining the severity an fatality of the illness.

5)The McFarland standard is a turbidity standard which is present as gold reference standard for the bacterial susensions being prepared. They are usually compared to 0.5 McFarland standard value and adjusted accordingly. For a turbidity standard value of 1 the cell density is approximately 3x10^8 CFU/mL. Since the number of organisms are too much, the results in the antimicrobial susceptibility test using the Kirby Bauerdisk diffusion method will be falsely sensitive.

6) The presence of colonies in the inner edge of the zone of inhibition clearly indicates that the micro-organism- Staphylococcus aureus is resistant to the action of the antibiotic- Oxacillin. The zone of inhibition clearly shows the antimicrobial action and efficiency of the antibiotic but the presence of the micro-organism talks about its resistance to the antibiotic.