Microbiology Lab Techniques

Question 1

Gram Stain Principles:

Why do gram positive bacteria turn purple?

Why do gram negative bacteria turn pink?

Answer 1

Once stained with crystal violet:

Their thick peptidoglycan cell wall allows them to resist alcohol decolourisation and they retain the dark purple colour.

Lack of peptidoglycan
Allows crystal violet to wash out and so decolourise
Decolourised bacteria can be counterstained pink with further application of a different dye - Safranin

Question 2

Technique:

How is a gram stain done?

Answer 2

(1) Primary stain - Crystal violet for 1 minute - rinse
(2) Stain with iodine - rinse
(3) Decolorisation with acetone - rinse
(4) Counter stain - safranin - rinse and blot

Question 3

Gram positive cocci:

Shape?

Shape and chain formation in the following?:

• Strep pneumoniae
• Enterococci
• Oral (Viridans)
• Strep progenies

Answer 3

Oval or round

Strep pneumoniae – PAIRS

Enterococci (a subset of Streptococci) – SHORT chains

Oral (viridans) Streptococci – LONG chains

Strep pyogenes – MEDIUM to LONG chains

Question 4

Agar plate:

Strep generally grow on an agar plate contained sheets blood.

How can you differentiate gram positive strep and staph?

Haemolysis:

Streptococci produce extracellular enzymes which lyse RBC’s in agar.

Colour could suggest what type of strep bacteria it is.

LOOK AT IMAGE.

Alpha haemolysis - partial - GREEN:
- Example? ***

Beta haemolysis - complete - CLEAR:
- Pyogenes

Gamma haemolysis - none - RED:
- Example?

Answer 4

Use a catalase test

Colony taken from agar plate - dipped in hydrogen peroxide

STAPH contains catalase - so bubbles seen as it converts hydrogen peroxide to H20 and O2

======

Alpha - green - strep pneumoniae

Gamma - red NO CHANGE - Entercoccus

Question 5

Further Identification:

Usually not needed as differentiating them doesn’t change medical treatment.

Usually needed for endocarditis so specific antibiotic susceptibility is known.

What 2 lab tests are used?

Answer 5

Mass spectrometry

Biochemical reactions

Mass spectrometry has become an important tool for the clinical microbiologist for bacterial identification. … Phage amplification (PA) has added new dimensions including sensitivity, specificity, and reduced culturing time to bacterial characterisation by MALDI-TOF-MS-based protein identification.

Biochemical reactions are very important in the identification of bacterial isolates and in the identification of different bacterial species. These tests depend on the presence of certain enzymes, such as catalase, oxidase, urease, gelatinase, etc., produced by the bacteria.

Question 6

Susceptibility Testing:

Disc testing - Describe it?

E tests - more specific:
- Describe it?

Both done in combination

Answer 6

The bacteria is reported as susceptible to that antibiotic

Filter paper soaked in specific concentrations of ABs
Placed on agar plate
Bacteria diluted in water

Zones of inhibition of bacterial growth around discs suggest the particular antibiotic kills the bacteria

MIC - minimum inhibitory concentration - the lower it is, the better the ABs