staining, cultures and other tests Flashcards
Gram staining - ‘Come In And Stain’ € Ziehl-Neelsen stain € Cultures; blood agar, XLD, MacConkey, CLED, Sabourard, Lowenstein-Jensen € Catalase test € Coagulase test € Alpha beta gamma haemolysis € Optochin test
how to carry out a gram stain
- apply crystal violet to heated fixed bacteria (heat by passing over Bunsen burner) and rinse with water
- add iodide, binds to the crystal violet
- decolourise with ethanol or acetone. wash off excess with water
- counterstain with pink safranin. rinse and dry
- observe if pink or purple
what colour are gram negative bacteria and why ?
thin peptidoglycan layer which does not retain crystal violet (has pores int it), but is stained by safranin, therefore appears pink
what colour are gram positive bacteria and why ?
Gram positive bacteria have a thick peptidoglycan layer which retains the crystal violet-iodine complex (thickness creates a barrier) therefore appears purple
what is the coagulase test used to identify ?
differentiating Staphylococcus aureus from other staphylococci
(staphylococci = gram positive and aerobic)
what does a positive coagulase test show?
which bacteria is present in coagulase positive
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
what does a negative coagulase test show?
which bacteria is present in coagulase negative?
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 )
what is the Catalase test used for?
to class gram positive cocci aerobic bacteria further as staphylococcus catalase positive or staphylococcus catalyse negative
what is the reaction happening during catalyse positive test
2H2O2 –> 2H2O + O2
what does the haemolytic test show?
what’s it used to classify ?
the ability of bacteria to break down RBCs in blood agar
classifies streptococci as beta-haemolytic,alpha or non
what does the oxidase test show?
Tests if micro-organism contains a cytochrome oxidase or indophenol oxidase
implies organism able to use oxygen as the terminal electron acceptor
what does a post oxidase test look like?
In oxidised state, indicator dark blue or maroon
why do we need to differentiate between gram positive and negative?
because they behave are treated differently according to if they are positive/negative
what colour does beta haemolytic streptococci make the agar plate go and why?
full Haemolysis - produces strong enzymes which completely break down blood (complete lysis) making the agar become….
transparent
what colour does alpha haemolytic streptococci make the agar plate go and why?
Partial haemolysis - goes green
this is because enzymes produced by bacteria only partially break down the RBCs on the blood agar
what colour does gamma haemolytic streptococci make the agar plate go and why?
lack of haemolysis
no change observed
the is Lancefield grouping needed?
for further categorising of strep
what is Lancefield grouping based on?
bacterial carbohydrate cell surface antigens
how is lancefild grouping carried out?
add antibodies to suspension
antibodies clump together like curdled milk
what is lancefild group A?
S.pyogenus
list some direct lab methods to diagnose bacteria infection
gram stain
acid fast stains
wet film
KOH for fungi
india ink
list some culture methods to diagnose bacterial infection
solid media
liquid media
blood culture
why does a gram stain only work for a fresh culture of bacteria?
after time cells lost ability to retain the stain therefore need to be fresh
give an example of a simple/basal media
when is this media used?
agar media , tryptone soy agar (TSA)
to grow non fastidious microorganisms
what is Enriched media
give example of an enriched media
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)
what is selective media
give example of an enriched media
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
what is Selective differential medium
give an example
how is it different from selective media ?
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)
what is Chromogenic media?
how does it work ?
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
give an example of a liquid culture media and for what type of bacteria its particulary useful for
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
what is Enzyme-linked immunosorbent assay?
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
what is the AFB test?
Acid fast bacilli – stain used to identify organisms with wax-like, thick cell walls e.g. mycobacteria (resistant to gram stain).
what is the Ziehl-neeslesn stain?
a rapid and practical method for detecting acid-fast bacilli (AFB).
what colour does mycobacteria stain with Ziehl-neeslesn stain?
red
give an example of a disease that we use Ziehl-neeslesn stain over gram stain and why
Use it to find TB because its a mycobacteria with thick lipid rich cell wall that won’t stain with gram stain
why is Use is Ziehl-neeslesn stain preferred in low income countries?
low cost and rapid
