Diagnostic bacteriology

Question 1

Difference in physical and chemical composition of Gram +ve and -ve bacteria

Answer 1

Gram-Negative = 2 outer membranes with thin peptidoglycan
Gram-Positive = 1 outer membrane with thick peptidoglycan

Question 2

What are the 3 main types of bacteriological tests?

Answer 2

1) To find type of bacteria: Microscopy - gram stain (gram +ve or -ve)
then can use morphology (spheres: coccus , rounded ended cyclinders: bacillus)
2) To find species of bacteria: Culture -Hemolysis test
Lactose test
Catalase test
Coagulase test
3) To choose treatment: Sensitivities -Disk diffusion
MICs
Break-points

Question 3

Why do gram +ve and -ve bacteria stain differently?

Answer 3

Bacteria are attached onto glass slide
Crystal violet solution folllowed by iodine solution is added to the bacteria –> Forms crystal violet and iodine complexes
Washed with alcohol –> Tightens peptidoglycan cell wall
Gram +ve: thick wall, complexes cannot be washed out
Gram -ve: thin wall, complexes are washed out
Counter stain with safranin: Gram +ve remain purple as are stained with crystal violet
Gram -ve: appear pink as have no crystal violet but are stained pink by safranin instead

Question 4

What are the 2 different types of culture that can be used to classify bacteria?

Answer 4

Culture on blood agar plates is a method to assess haemolytic activity. It is useful to further classify Gram-positive bacterial species.

Culture on plates containing lactose is a method to assess lactose fermentation. It is useful to further classify Gram-negative bacterial species.

Question 5

How is bacteria hameolytic activity measured with culture?

Answer 5

Pick a bacterial
colony
Streak out the bacterial
colony onto blood agar containing 5% sheep RBC
Incubate overnight and assess haemolytic activity next day:
Gamma-hemolysis = no hemolysis: produce no hemolysins (enzymes that damage RBC) (no zone)
Alpha-hemolysis = partial hemolysis (opaque zone)
Beta-hemolysis = complete hemolysis (transparent zone)

Question 6

How is bacteria lactose fermentation assessed with culture?

Answer 6

Streak bacterial colony onto MacConkey agar containing bile salts, crystal violet
and lactose> Incubate overnight and assess next day. Lactose fermenting = bacteria produce lactic acid during lactose fermentation –> PINK COLONIES
Lactose non-fermenting = bacteria do not ferment lactose –> COLOURLESS COLONIES

Question 7

What are the 2 main enzyme tests for deciding bacteria species?

Answer 7

The catalase test is a method to assess catalase activity. It is useful to further classify Gram-positive bacterial species. It can discriminate staphylococci (produce catalase) and streptococci (do not produce catalase).
Catalase + = O2 bubbles.
Catalase - = no O2 bubbles.
The coagulase test is a method to assess coagulation of plasma. It is useful to further classify Staphylococcal species. It can discriminate S. aureus (produce coagulase) and other staphylococci (do not produce coagulase).
Coagulase + = clumps in plasma.
Coagulase - = no clumps in plasma.