Question

Question 1:
Why a pure colony isolation on the plate is necessary?
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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
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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?

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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?
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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)

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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

Answer 1:

It is essential to get a pure colony isolated on solid media in order to study the growth pattern and biochemical reactions of the organism and give it a positive identity. As bacteria vary a lot in these characters from organism to organism and also lots of variations are seen within the same organism species, it is essential to have the pure growth colony which is practically arising from a single bacterium when the isolation technique is properly applied.

Answer 2:

There is need to get more from the primary culture than just a single isolated colony. In order to achieve the final goal, following things along with primary culture help:

1. An Enrichment Broth - is used when isolating a pathogenic organism from a clinical specimen which otherwise has a bioload with normal flora. Best example is use of Selenite F broth to enrich pathogens (Salmonella / Shigella) in stool which is full of normal flora coliforms. The enrichment broth inhibits the normal flora to some extent giving chance to pathogens to multiply more and become visible when isolation is done from this medium
2. Nutrition Agar - this basal medium is nutritive in nature and supports growth of maximum organisms. This is useful when the microbial load is expected to be too low and hence no inhibitors should be there in the medium so as to get at least few colonies of the organism of interest. This is more useful for getting bacteria to grow if they are present from specimen which would otherwise be sterile. For example, sing Sheep Blood Agar Plate to culture body fluids like CSF / Synovial fluid etc.
3. Selective Media - they are as the name suggest, selective for one group of organisms while suppress the growth of others. Use of antibiotics infused agar mediums to suppress the commensals to get growth of one group of microorganisms that we know are not affected by the presence of this antibiotic. For example using Chloramphenicol Agar to grow fungi and yeast while suppressing bacteria from growing.
4. Differential media - are essentially part of the isolation process as they differentiate between two groups of organisms, taking first step towards identification. Commonest medium is this category is MacConkey's Agar which helps differentiate between Lactose Fermenters (pink colonies) from Lactose non-fermenters (pale colonies) even as we isolate the organisms on this medium.

Answer 3:

If the slide smear is made too thick with organisms we will miss the organisms that are less in number as the one with more numbers will mask them and make them invisible for us. Also, with too many bacteria on the slide, the morphological characters will be difficult to discern, e.g.. getting confused between Streptococci and Staphylococci on thick smear. Similarly, making the smear too thin by placing less organisms on the slide, we will find it difficult to focus ans see the organism within the visual field and will have to spend longer time searching for it on the slide.

Answer 4:

The term antibody titre refers to that dilution of the antibody below which the agglutination will not happen. It is an indicative of the antibodies level in the serum. This is very easy and reproducible result of immunological testing and helps us identify and quantify (semi-quantitative) the infection status. For example - Antibody titre of 1 : 80 is considered positive for Salmonella typhi, in Widal test and confirms diagnosis of the patient even if we failed to grow the pathogen from blood or stool samples directly.

Paired sera is tested where the results expected have not been reached to. Explaining with example - if on admission a patient having all signs and symptoms of Typhoid fever gave a Widal test negative will be asked to repeat the test after a certain period (5 - 7 days) to see if the titre is increasing over time and confirming the diagnosis. Also, it is useful where we know an active infection is happening but are unable to demonstrate presence of IgM antibodies; then we do paired sera for Ig G antibodies, one week apart to show increase in titre of ig G antibodies.

Answer 5:

The McFarland Turbidity Standard 0.5 is commonly used to match turbidity of bacterial growth suspension for carrying out antimicrobial susceptibility test. Now, if the turbidity is matched with Std 1 instead of Std 0.5, then we are inoculating with higher number of bacteria. This will give a heavy growth on the medium, resulting in False Resistance report whereas organism will actually be sensitive. This happens so because Kirby Baeur Disc Diffusion depends upon MIC of the drug and will be correct only when a known standard cfu / ml is used.

Answer 6:

While checking Staphylococcus aureus susceptibility to Oxacillin disc, if we observe a zone of inhibition corresponding to sensitive organism, yet we see the tiny colonies in the zone of inhibition, we will report it as Resistant to oxacillin. This is because, the tiny colonies represent the resistant mutant in the population and if we give this drug for treatment, we will end up removing the sensitive organism and selecting the resistant mutant which will keep the infection going and eventually we will have to change the antibiotic. In order to not selectively support the resistant mutant, we must report Staphylococcus aureus as resistant to Oxacillin in the very first go.