Lab related SAQs Flashcards
Consider the catalase test. Briefly explain why the formation of bubbles are indicative of a positive result
Catalase in an enzyme which breaks down hydrogen peroxide which is toxic to the cell, forming oxygen gas as a result. The bubbles formed during the test indicates that hydrogen peroxide is being broken down and oxygen gas is released hence is indicative of a positive result.
Consider the oxidase test. What would indicate a positive result? Explain
The oxidase test is used to identify bacteria that produce cytochrome c oxidase, an enzyme of the bacterial electron transport chain. (note: All bacteria that are oxidase positive are aerobic, and can use oxygen as a terminal electron acceptor in respiration. This does NOT mean that they are strict aerobes. Colour change pink → purple → black
Would you expect a strict anaerobe to be oxidase positive or negative? Explain.
A strict anaerobe would be negative, as the oxidase test is only positive for aerobes.
Consider the indole test. What would indicate a positive result? Explain
The indole test is a biochemical test performed on bacterial species to determine the ability of the organism to convert tryptophan into the indole. This division is performed by a chain of a number of different intracellular enzymes, a system generally referred to as “tryptophanase.” A positive result would indicate a pink colour change.
Describe an identification technique used in lab
Gram staining → gram-positive or gram-negative
16s metagenome analysis - Illumina
Sanger gene analysis → composition of the DNA allowing comparison to known DNA strands of different types of microbes
Biochemical test → catalase, oxidase, indole
Is Luria broth defined or complex? Name one of the carbon and energy sources in the media
Luria broth contains yeast extracts which is undefined hence the broth is complex.
Carbon source is the amino acids provided by tryptone.
For the three individual components state whether they are defined or undefined.
Tryptone; Yeast extract and NaCl.
Tryptone - defined
Yeast extract - undefined
NaCl - defined
What is the media component that makes McConkey Agar selective. Explain.
The crystal violets in the media prevents gram-positive bacteria to grow on the plate hence making it selective to gram-negative bacteria.
What is the media component that makes McConkey Agar selective and/or differential. Explain.
Contains bile salts that inhibit many non - enteric organisms making it a selective medium for gram -ve. It also contains the carbohydrate lactose and the pH indicator neutral red, allowing differentiation between lactose fermenting and nonfermenting lineages. This makes it an indicator or differential medium.
What microorganisms does Sabouraud’s agar select. Explain.
Contains a high concentration of glucose at a low pH that inhibits bacteria. Used for the isolation of yeast and fungi.
List and give the function for each of the components that define Mannitol Salt Agar as a selective and differential medium
High concentrations (7.5-10%) of NaCl → selective to gram-positive bacteria eg Staphylococcus and Micrococcaceae
Mannitol → differentiate between mannitol fermenting staphylococci
pH indicator is present to show if fermentation of mannitol occurs
If the agar turns red → acidic which means fermentation of mannitol is occurring
Predict what growth would be seen on a MSA plate inoculated with S.aureus and incubated anaerobically.
S.aureus is a facultative anaerobe hence when incubated anaerobically it is expected for the MSA plate to turn a red/pink colour as fermentation of mannitol occurs.
What is the media component that makes Horse blood agar an enriched agar? Explain
The red blood cells from the horse blood makes the agar enriched as it supports the growth of most pathogenic bacteria and yeast.
What is the media component that makes Horse blood agar differential? Explain
The reaction of -haemolysis (complete haemolysis → clear zones around the bacterial colonies) and -haemolysis (partial haemolysis → greenish zones around the colonies) differentiates the bacterial isolates depending on it’s haemolytic effect.
Briefly describe the process of gram staining. What dyes are involved?
After sterilising the loop, take a loopful of a single colony and place it on a microscope slide. Pass the slide through the bunsen burner flame so that it sticks to the slide before staining. Place 2-3 drops of crystal violet onto the slide covering the sample and leave for a minute, then wash using deionised water. Add concentrated iodine and allow to stand for approximately 1.5minutes and wash well with water. Then decolourise with alcohol iodine reagent until violet colour ceases to run and wash well. Then the counterstain carbol fuchin is added for 15-20 seconds before washing off and allowing the sample to dry before looking under the microscope.
Dyes:
Crystal violet
Carbol fuchin
