Lecture 7 Diagnostic Microbiology in the Clinical Setting

Question 1

What is the mechanism of the gram stain? (hint: cell wall)

Answer 1

G+ bacteria have peptidoglycan in cell wall which will retain crystal violet-iodine complex, so retain purple when decolourised.
Whereas G- bacteria have lipopolysaccharides on outer membrane without a peptidoglycan cell wall so crystal violet-iodine complex is decolourised and stains red by counterstain safranin

Question 2

Looking as at a gram stain, how can you be sure that the stain is working? (which cell acts as a control)

Answer 2

Neutrophil from pus appears pink

Question 3

Define acid-fast organism.

Answer 3

Acid-fast organisms have an impermeable cell wall with mycolic acid which retain acid-fast stains even after decolorisation e.g. Mycobacterium. Look red on blue background.

Question 4

Name an acid-fast stain.

Answer 4

Ziehl Neelsen

Question 5

What does iron haemotoxylin detect?

Answer 5

Fecal protozoa (parasite)

Question 6

What is Sabourauds dextrose media?

Answer 6

Consists digests of casein and animal tissue supplemented with glucose.

Question 7

What is a chromogen plate?

Answer 7

Contains chromogenic substrates which are specifically cleaved by bacterial enzymes, resulting in bright colours characteristic of a bacteria

Question 8

Describe the mechanism of Lancefield group agglutination test for Streptococcus.

Answer 8

A solution of different latex beads coated with antibody of the specific Lancefield group is put into each of the wells, which will recognise specific antigen carbohydrate on Streptococcus. Positive result is agglutination.

Question 9

Which type of haemolysis and Lancefield grouping is Streptococcus pneumoniae?

Answer 9

alpha-haemolysis

Not groupable for Lancefield grouping

Question 10

Which type of haemolysis and Lancefield grouping is Streptococcus pyogenes?

Answer 10

beta-haemolysis

Lancefield Group A

Question 11

What are the benefits of automated blood culturing gin terms of quantifying results?

Answer 11

Base of bottle changes colour which can be detected and quantified by sophisticated light detection

Question 12

What is the purpose of lytic agents in blood cultures?

Answer 12

Breakdown WBCs so bacteria has greater growth, this reduces growth time.

Question 13

How does MGIT (Mycobacteria Growth Indicator Tube) work?

Answer 13

At the bottom of tube is fluorescent compound embedded in silicon. Initially when the tube has high oxygen, quenches emissions from the fluorescent compound. As bacteria grows, oxygen is being used up, so fluorescent emissions shine through.

Question 14

In antibiotic sensitivity tests using tube broth dilutions, which tube is the MIC?

Answer 14

First tube that is clear and without growth (lowest conc of antibiotic that inhibits bacterial growth)

Question 15

How does MALDI-TOF work?

Answer 15

Sample is co-crystallised with the matrix on a target plate. Use a pulsed laser light of UV or ER, creates a scatter profile detected by mass spectrometer.

Question 16

What is digital PCR and when is it used?

Answer 16

When the sample is a heterogeneous mixture of DNA molecules and the target molecules is at very low levels. Need to split this into smaller units where there is 1 copy of DNA in each unit, this enriches minority targets.

Question 17

What are microarrays?

Answer 17

DNA chip array containing DNA fragment probes attached to a solid surface, will hybridise with target DNA fragment which can be detects using fluorescence