Lecture 3 - Microbial diagnostic Flashcards
what is the general process to identify what microorganism caused a disease?
1: find the culprit (swabs, blood, urine)
2: grow the culprit (agar, liquid medium, animal cells)
3: identify the culprit (array of diagnostic methods)
what is a colony of bacteria?
where one single bacterium was isolated on an agar plate and grew logarithmically overnight to produce millions of cells. we see a dot or shape about the size of 1-10mm
what is a cocci?
round bacterium
give an overview of diagnostic methods
- structural features (colony and cell morphology)
- biochem properties (O2 depen, AB and chem resistance)
- antibody binding (serological tests, immunological assays
- infection by phage (phage typing)
- DNA/RNA (hybridisation and PCR)
what can we tell by the colony morphology on an agar plate?
gives some clues but isn’t sufficient enough for conclusive identification
how can colony morphology differ?
By:
- shape
- colour
- appearance
- odor
which gram type bacteria stains purple and why?
gram positive
- the crystals of crystal violet get trapped in the thick peptidoglycan wall
why doesn’t gram negative stain purple?
even though it has a peptidoglycan wall, it isn’t thick enough to retain crystals, and wash off when acetone is used during decolorisation
how can we identify gram negative bacteria?
by doing a counter stain of safranin
true or false: candidas albicans retains crystal violet, and is therefore a gram positive bacterium.
false, candidas albicans does retain crystal violet but is a yeast, so is neither gram -ve nor gram +ve.
how does the cell size of candidas albicans relate to bacteria?
significantly larger than bacteria
what is budding in yeast?
a form of asexual reproduction, a bud receives cytoplasm and organelles before detaching when it matures into its own cell
what are pseudohyphae?
cells that began to bud but ‘got stuck’ and instead remain on the parent cell as a chain-like structure
what is an acid-fast bacterium/mycobacterium?
Bacterium which have waxy, hydrophobic coat made of mycolic acid
what kind of cell wall morphology do mycobacterium have?
gram positive -> they still contain the thick peptidoglycan cell wall.
why can’t we use normal gram-staining methods on mycobacterium?
because their thick waxy coat is hydrophobic, and most stains are hydrophillic
briefly describe Ziel-Neelson (acid fast) staining
1: carbolfuchsin (red) and heat are applied to breach waxy coat of mycobacterium
2: ethanol and HCl added to destain (epithelial cells decolour, acid-fast cells retain colour)
3: methylene blue is added as a counterstain to stain other cells.
after completing a Zeil-Neelson stain, what would you expect to see in the tissue?
acid-fast (myco) bacterium stained red, with all other cells stained blue
what stain do we use to visualise moulds such as penicillium?
Lactophenol cotton blue stain
how does lactophenol cotton blue stain work?
1: phenol kills the mould and prevents cell lysis
2: cotton blue stains the chitin in the cell wall
what are structures 1,2 and 3?
1: Hypha
2: fruiting body
3: spores
what are the three assays we can do to test for different enzyme activity?
1: catalase test
2: coagulase test
3: oxidase test
what does the catalase test involve?
placing two types of bacteria on a microscope slide containing a spot of hydrogen peroxide. If the sample bubbles, catalase is present
what does the presence of catalase tell us about the bacterium?
it is staphylococci
what does the coagulase test involve?
- suspend a bacterial colony in rabbit plasma.
- if a coagulant is formed after 1-4h (fibrinogen -> fibrin) then coagulase is present
what does the presence of coagulase tell us about the bacterium?
Staphylococcus Aureus is present
(no other staphs have coagulase)
Coagulase is a ____ ________-_____ protein that mediates fibrin polymerisation
cell surface-bound
what does the oxidase test involve?
a sample of bacteria is placed onto a window containing an oxidase reagent.
- if the sample turns blue, then oxidase is present
what are some oxidase positive bacteria?
- pseudomonas
- neisseria
- moraxella
- campylobacter
Microbact strips are an assay based on?
metabolic reactions
what does a microbact strip assay involve?
- mini test systems which use standard biochemical identifications
- each position on the strip is an independent reaction
- test is for a specific enzyme, substrate or product
- indicator is added to monitor biochemical reaction
what was the example of a test reaction in microbact strip testing we discussed in class?
testing for fermentation of glucose when bacteria is added:
- since fermentation produces acid, pH drops and [H+] rises.
- if glucose fermentation is present, the indicator in the glucose sample will change colours.
- if there is no fermentation, the glucose will remain blue.
how do you use a microbact analysis sheet?
the strip is split into (typically 4) groups of three.
- the first well of each 3 has an index of 4
- the 2nd well has an index of 2
- the 3rd well has an index of 1
add the index numbers for each positive result (compared to a reference microbact chart) per group of three.
- this will give you a 4 digit code which corresponds to a particular bacterium
what is selective agar?
agar that contains inhibitors to prevent growth of certain organisms
what is differential agar?
contains indicators to differentiate organisms
- cause visible changes in response to bacteria metabolism
what is sabourad agar?
agar that is selective for fungi, but isnt differential. the low pH suppresses the growth of most bacteria
what is Eosin-methylen blu agar (EMB)?
- selective for gram -ve bacteria
- contains aniline dyes which are toxic to gram positive bacteria
- also is differential -> differentiates lactose fermenters
what is MacConkey agar?
- selective for intestinal pathogens
- bile salts inhibits non-enteric bacteria (bacteria not in the enteric system)
- also differentiates lactose fermenters (go pink on orange agar)
what is blood agar?
- non selective but differentiates types of heamolytic reactions
- grows many types of fastidious bacteria
describe how beta, alpha and gamma heamolysis reactions would look on a blood agar plate
alpha: partial hemolysis
beta: full hemolysis
gamma: no hemolysis
what is mannitol salt agar?
selective for haloduric bacteria (staphyl)
- differentiates mannitol fermenters (yellow on pink plate)
- Staph aureus is the only staph that ferments mannitol
what does haloduric mean?
can endure higher than normal salt concentrations
what is bile-esculine agar?
- selective for enteric bacteria.
- oxgall (a bile salt) inhibits non-enteric bacteria
- esculine hydrolysis is performed by enteric bacteria, so if they are present, then the agar turns black
what is a bacterial lawn?
an agar plate that has been completely covered in a strain of bacteria
what will the bacterial lawn look like if an antibiotic is placed in it that the bacteria are sensitive to?
there will be a thick ring around the AB of no bacterial growth
true or false, the antibiotic susceptibility test is selective for certain bacterium
false, it allows for differentiation between species.
- e.g strep p is sensitive to bacitracin, but strep d is resistant.
what is agglutination
the action of an antibody when it cross-links multiple antigens producing clumps of antigens
what is cell agglutination most commonly used to distinguish?
whether or not a specific bacterium serotype is present.
how does cell agglutination work?
by knowing that a specific antibody will bind to a specific antigen on a serotype’s surface, we can put the antibody in the unknown bacterium sample and if it clumps, then we know that antigen is present.
what is latex bead agglutination?
the same concept as cell agglutination, only the antibodies are attached to a latex bead, which makes the interaction easier to visualise.
what is a western blot?
- separation of proteins by size using electrophoresis is washed by a primary antibody
- then it is washed with a secondary antibody and FastRed stain, which binds to the 1º antibody
- one protein on the chart will be red.
what does a western blot help us find out?
identification of a specific protein
or identification of an antibody in serum using specific proteins
what do all methods of distinguishing through antibody-antigen binding have in common?
they are all highly specific
What is ELISA?
similar to western blot, but the reaction is in a solution
- e.g using a protein that binds an antibody or vice versa
what is a benefit of ELISA over western blot?
it is easier to quantify, e.g more colour = more protein/antibody
where can we commonly see a lateral flow test
in a covid or pregnancy test
what concept does a lateral flow test use?
antibody antigen binding
what is immunofluorescence microscopy?
- fluorescently labelled antibodies are either directly or indirectly (bind to 2º AB) added to a solution and helps show location of specific proteins in a cell or tissue
what does RFLP differentiate?
strains of the same species
how does RFLP work?
a mutation may cause a strain to destroy a restriction site, causing one less and therefore a longer fragment on gel electrophoresis
- differentiates mutant strain from og
what does RFLP stand for?
restriction fragment length polymorphism
what is RFLP mainly used for?
epidemiological studies, but used to be used for DNA fingerprinting
what is PCR used for?
growing samples to a big enough visual size
- means we can visualise microorganisms that do not grow in culture, such as chlamydia and viruses
how does PCR work?
- heat stable DNA polymerase creates copies of DNA with the help of initial heat denaturing the protein and an oligo DNA primer to start DNA polymerase off
why can’t chlamydia be grown in culture?
it relies on mammalian cells to replicate and grow
- grows inside of them
what is FISH?
uses fluorescent probes to target specific DNA or RNA sequences
- 16S RNA is a common target
