what is the role of the veterinary technician in microbiology
Isolate the pathogen, identify the pathogen, determine which antibiotics will effectively kill the pathogen, report finding to the veterinarian/ clinic.
what is the sequence of tests for microbial specimens
- collect sample
- gram stain (microscopic exam)
- inoculate culture media
- incubate 18 - 24 hours at 37 degrees celsius
What are the 5 types of bacteriological media we can use
agar, broth, plate, tube, slant
what are the 6 types of culture media
transport, nutritive, selective, differential, enriched, enrichment
what is transport culture media?
keeps microorganisms alive; no growth
what is nutritive (general purpose) culture media?
growth of all types of bacteria and some fungi e.g. chocolate agar, blood agar
what is selective culture media?
contain compounds that inhibit growth of certain types
organisms; selects for certain bacteria
what is differential culture media?
differentiates between certain types of bacteria based on biochemical reactions e.g. lactose fermenters
what is enriched culture media?
addition of blood, serum or egg
what is enrichment culture media?
liquid media favours growth of certain bacteria
what must all media be checked for?
accidental contamination
what are some gram-positive culture media plates?
Columbia collistin-naladixic acid agar (CNA); Phenylethyl alcohol agar (PEA)
what are some gram-negative culture media plates?
MacConkey (MAC), Eosin methylene blue (EMB), Salmonella-Shigella agar (SS) and Bismuth Sulfite agar
What is important about Blood agar plates or trypticase
soy agar?
5% sheeps blood
-differential )allows hemolytic reaction to be seen)
What is alpha hemolysis?
incomplete hemolysis; narrow greenish or slimy band around bacterial colony
What is beta hemolysis?
complete hemolysis; no change in appearance of agar around colony
what is delta hemolysis?
double zone of hemolysis
what is uricult?
a combination/modular media
good for urine
what structure allows bacteria to be motile?
flagellum
what are motility test medium used for?
used to show bacterial motility
motility: growth throughout medium
no motility: growth along stab line only
what are 9 tests used for identification of bacteria?
gram stain, acid fast stain, potassium hydroxide, catalase, coagulase, kirby-bauer sensitivity, optonchin susceptibility, oxidase, CAMP
If you don’t have a gram stain what test can u use?
KOH test
what is the KOH test? how does it work?
it is a general test for differentiation of Gram + and Gram - bacteria
- 3% KOH added to a colony on a glass slide, mix for 2 min, if you get a mucoid appearance and a sticky strand = gram postitive, if no change = gram negative
what is the catalase test? how does it work?
- finds gram+ cocci and small gram + rods
- wooden applicator stick used to smear small amount of colony to be tested onto slide, a drop of 3% hydrogen peroxide
- bubbling = postive reaction
what genus are positive on the catalase test?
Staphylococcus spp.
what genus are negative on the catalase test?
Streptococcus spp.
What test can be also used for Staphylococcus spp.
Coagulase test
how does the coagulase test work?
a sample of Staphylococcus colony placed in small amount of rabbit plasma with EDTA, incubate overnight
- if plasma gels= coagulase - positive Staphylococcus spp = S. aureus
- if plasma remains liquid = coagulase-negative = S.epidermidis
what is the lancefield grouping based on?
antigenic difference in cell wall carbohydrates
what is the lancefield grouping used for?
used for group catalase-negative and coagulase-negative bacteria
what are the ten tubes of broth and agar
trpyricase soy broth, brain-heart infusion, thioglycollate broth, gram-negative broth, urea agar slant, bile esculin again, simmon’s citrate tubes, triple sugar iron agar, sodium chloride 0.85%, motility test medium
what is trypticase soy broth used for?
general use, grows most bacteria (fastidious bacteria especially), blood culture, sterility testing
what is brain heart infusion used for?
-enriched broth: used to bring bacteria to certain turbidity level for diffusion antibiotic sensitivity testing
what is the brain heart infusion agar used for?
used to determine oxygen requirements of bacteria
what is the thioglycollate broth used for?
grows most bacteria, anaerobic bacteria and some fungi, blood culture, can also be used to determine oxygen requirements for bacteria
what are gram-negative broth used for?
used in fecal culturing-enrichment medium for growing Samonella spp. and Shigella spp.
What is urea broth agar slant used for?
determines whether bacteria produce urease
-the ammonia reacts with the pH indicator phenol red, turing the broth a hot pink color
what does urease degrade into?
it degrades urea into ammonia, water, and CO2
What are Bile esculin agar tubes used for?
primarily used to differentiate Streptococcus spp from Enterococcus spp.
A positive reaction = ferric citrate in agar turns dark brown to black
-bile salts are selective (Gram + inhibited)
What are simmons citrate tubes (slant) used for?
differentiates bacteria according to their use of citrate
- often used to differentiate Gram negative bacteria
- postive result - deep blue color
- unchanged medium = green
what does the sodium chloride 0.85% test do?
-sterile solution used for diluting Gram negative bacteria for API20E
What does triple sugar iron agar do?
classifies bacteria based on ability to ferment glucose, lactose and sucrose
what does yellow mean in triple sugar iron agar?
fermentation of carbohydrates produces acid end products and changes phenol red indictor to yellow
what is the ph indicator color for triple sugar iron agar?
phenol red
what also can triple sugar iron agar classify?
bacteria based on hydrogen sulfide production (blackening) and gas production (bubbles and cracks)
What are some musts of Antibiotic discs?
- must be kept at 4 degrees C
- must not be expired
- must be monitored using controlled organisms
- drug concentration = therapeutic levels in animals
- must be 10 to 15 mm from the plate edge
what is direct sensitivity testing?
- application of undiluted samples (urine)
- precision increased when only 1 organism present
For indirect sensitivity testing what degrees must it be incubated overnight at?
37 degrees C
How often do you read the Kirby-Bauer sensitivity test?
18 to 24 hrs
What is the Kirby-Bauer sensitivity test?
the Mueller-Hinton agar with blood - zones read on top without plate lid
-results can be susceptible,resistant, or intermediate
What is the Optochin susceptibility used to detect?
Streptococcus pneumoniae
Give an example of a bacteria that using an Oxidase test will change to a dark blue colour after 5 minutes?
Pseudomonas aeruginosa
What is the Mueller-Hinton agar culture media plate used for?
general-use medium specifically formulated to give standardized results during antibiotic sensitivity testing (Kirby-Bauer test)
For Mueller-Hinton agar culture media plates what can they be enriched with for more fastidious organisms
blood
What to Salmonella-Shigella agar plates select for?
gram-negative bacteria
What do Salmonella-Shigella agar plates differentiate?
- colonies on the basis of lactose fermentation (pink)
- differentiate hydrogen sulfide-producing bacteria by the use of ferric citrate in the formula
What is a coliform bacteria and example?
E.coli
-ferment the lactose = bacterial colonies are always pink
What do Salmonella spp produce? ferment lactose?
they do not ferment lactose but produce hydrogen sulfide = bacterial colonies are colorless with black centers
What do Shigella spp. produce?
do not ferment lactose or produce hydrogen sulfide = colorless colonies
What do the Bismuth Sulfite Agar culture media plates do?
- supresses growth of coliforms (E.coli)
- allow growth for Salmonella spp.
- contains sulfur
What does it mean for a media to be modular/combination
- chambered agar plates with a variety of selective and non-selective agar
- enterotubes with a variety of media for enteric bacteria
Describe the Mannitol Salt Agar? use? about it? is it used often?
- not used routinely
- 7.5% sodium chloride with selects for halotolerant organisms (Staphylococcus spp.)
- Mannitol and phenol red (pH indicator) differentiate between organiusms which can ferment mannitol and those that cannot
What does the PEA agar stand for and what does it select for?
Phenylethyl alcohol agar, selection = gram postitive
What does CNA agar stand for? and what does it select for?
Columbia colistin-nalidixic acid with 5% sheeps blood
-select for gram positive organisms
Describe the Eosin Methylene Blue culture media plate?
-selects for gram negative bacteria
-differentiates between lactose and non-lactose fermentators
-strong acid production = metallic green (E.coli)
-weaker fermentation of lactose = pinkish purple
non lactose = colorless
what does the MacConkey ll agar plate select, and what does it use for it? What inhibits the growth of gram positive bacteria.
- the bile salts select for lactose-fermenting
- crystal violet inhibits growth of gram-positive
indicators: lactose and phenol red