staining, cultures and other tests

Question 1

how to carry out a gram stain

Answer 1

1. apply crystal violet to heated fixed bacteria (heat by passing over Bunsen burner) and rinse with water
2. add iodide, binds to the crystal violet
3. decolourise with ethanol or acetone. wash off excess with water
4. counterstain with pink safranin. rinse and dry
5. observe if pink or purple

Question 2

what colour are gram negative bacteria and why ?

Answer 2

thin peptidoglycan layer which does not retain crystal violet (has pores int it), but is stained by safranin, therefore appears pink

Question 3

what colour are gram positive bacteria and why ?

Answer 3

Gram positive bacteria have a thick peptidoglycan layer which retains the crystal violet-iodine complex (thickness creates a barrier) therefore appears purple

Question 4

what is the coagulase test used to identify ?

Answer 4

differentiating Staphylococcus aureus from other staphylococci

(staphylococci = gram positive and aerobic)

Question 5

what does a positive coagulase test show?

which bacteria is present in coagulase positive

Answer 5

Coagulase activates prothrombin to convert fibrinogen to fibrin
ie. a clot to form

therefore in positive test you can see a cloudy, clumpy clot showing coagulase must be present

S.aureus present

Question 6

what does a negative coagulase test show?

which bacteria is present in coagulase negative?

Answer 6

Coagulase activates prothrombin to convert fibrinogen to fibrin
ie. a clot to form

therefore in negative test, no clot forms, test tube is clearer and more liquid

no coagulase present means bacteria is a stpahylocci (but no S.aureus )

Question 7

what is the Catalase test used for?

Answer 7

to class gram positive cocci aerobic bacteria further as staphylococcus catalase positive or staphylococcus catalyse negative

Question 8

what is the reaction happening during catalyse positive test

Answer 8

2H2O2 –> 2H2O + O2

Question 9

what does the haemolytic test show?

what’s it used to classify ?

Answer 9

the ability of bacteria to break down RBCs in blood agar

classifies streptococci as beta-haemolytic,alpha or non

Question 10

what does the oxidase test show?

Answer 10

Tests if micro-organism contains a cytochrome oxidase or indophenol oxidase

implies organism able to use oxygen as the terminal electron acceptor

Question 11

what does a post oxidase test look like?

Answer 11

In oxidised state, indicator dark blue or maroon

Question 12

why do we need to differentiate between gram positive and negative?

Answer 12

because they behave are treated differently according to if they are positive/negative

Question 13

what colour does beta haemolytic streptococci make the agar plate go and why?

Answer 13

full Haemolysis - produces strong enzymes which completely break down blood (complete lysis) making the agar become….

transparent

Question 14

what colour does alpha haemolytic streptococci make the agar plate go and why?

Answer 14

Partial haemolysis - goes green

this is because enzymes produced by bacteria only partially break down the RBCs on the blood agar

Question 15

what colour does gamma haemolytic streptococci make the agar plate go and why?

Answer 15

lack of haemolysis

no change observed

Question 16

the is Lancefield grouping needed?

Answer 16

for further categorising of strep

Question 17

what is Lancefield grouping based on?

Answer 17

bacterial carbohydrate cell surface antigens

Question 18

how is lancefild grouping carried out?

Answer 18

add antibodies to suspension

antibodies clump together like curdled milk

Question 19

what is lancefild group A?

Answer 19

S.pyogenus

Question 20

list some direct lab methods to diagnose bacteria infection

Answer 20

gram stain

acid fast stains

wet film

KOH for fungi

india ink

Question 21

list some culture methods to diagnose bacterial infection

Answer 21

solid media

liquid media

blood culture

Question 22

why does a gram stain only work for a fresh culture of bacteria?

Answer 22

after time cells lost ability to retain the stain therefore need to be fresh

Question 23

give an example of a simple/basal media

when is this media used?

Answer 23

agar media , tryptone soy agar (TSA)

to grow non fastidious microorganisms

Question 24

what is Enriched media

give example of an enriched media

Answer 24

Contains basal media enriched with a substance to provide extra nutritions to allow growth of more fastidious microorganisms

Eg. blood agar media contains sheep blood, or chocolate agar (heated blood agar, RBCs have released their contents)

Question 25

what is selective media

give example of an enriched media

Answer 25

Help to differentiate between normal non-harmful flora and pathogenic microorganisms
Selective media contains something that inhibits the growth of harmless bacteria

eg. campylobacter Agar → contains antibiotics that prevent growth of unwanted microorganisms, have to know beforehand that the bacteria we want to grow is resistant to the antibiotics

Question 26

what is Selective differential medium

give an example

how is it different from selective media ?

Answer 26

Selective because it helps to differentiate between normal non-harmful flora and pathogenic microorganisms
But is also differential because it shows bacteria with different metabolic properties apart

eg. MacConkey agar →
Contains bile salts that inhibits normal GI bacteria growth, but allows harmful GI bacteria to grow eg. salmonella.
But also differentiates between species that ferment the sugar lactose and those who can’t (makes it differential)

Question 27

what is Chromogenic media?

how does it work ?

Answer 27

Use if we know a specific metabolic characteristic of the microorganism of interest

Multiple modified substrates are incorporated into the media . They are modified with the addition of a chromagin, a molecule that makes it produce a colour when its cleaved

Therefore if microorganism is present and has this specific substrate we were looking for, it will produce a colony of the corresponding colour

Question 28

give an example of a liquid culture media and for what type of bacteria its particulary useful for

Answer 28

Robertsons cooked meat

Allows growth of fastidious microorganisms
Patient sample is inoculated into cooked meat, incubated to allow any bacteria to grow for 24 hours
Portion is subcultured onto a solid media (eg. blood agar)

Technique helps to multiply bacteria, and is especially useful for anaerobic bacteria

Question 29

what is Enzyme-linked immunosorbent assay?

Answer 29

ELISA

format to measure specific serum antibodies

cover inside of wells with antigen of interest, fill wells with patient serum and if antibodies are present will bind to and fix to bottom of well

use a colergeni enzyme to show presence with colours

Question 30

what is the AFB test?

Answer 30

Acid fast bacilli – stain used to identify organisms with wax-like, thick cell walls e.g. mycobacteria (resistant to gram stain).

Question 31

what is the Ziehl-neeslesn stain?

Answer 31

a rapid and practical method for detecting acid-fast bacilli (AFB).

Question 32

what colour does mycobacteria stain with Ziehl-neeslesn stain?

Answer 32

red

Question 33

give an example of a disease that we use Ziehl-neeslesn stain over gram stain and why

Answer 33

Use it to find TB because its a mycobacteria with thick lipid rich cell wall that won’t stain with gram stain

Question 34

why is Use is Ziehl-neeslesn stain preferred in low income countries?

Answer 34

low cost and rapid