7. Bacterial Growth and Identification Flashcards
The name ‘staphylococcus’ comes from where? What does this mean?
- grape in Greek
- under a microscope appears as clumps of round cells, like a bunch of grapes
Where is Staph.aureus found in body?
- nose/anterior nares
- throat/nasopharynx
- skin
How do staph survive in body?
- withstand high concs of salt
- survive in sweaty areas
- provide a means of selectively culturing staph using high salt medium
What kind of bacteria is staph?
Exception?
commensal
- staph. aureus involved in infections and called opportunistic
What kinds of conditions are caused by staph aureus?
- broad range
- toxin-mediated conditions like toxic shock syndrome or food poisoning (preformed enterotoxins)
- localised skin infections like impertigo
- wound infections
- systemic infections like infective endocarditis
MRSA is what?
methicillin resistant staph aureus
Staph is gram …, … positive cocci.
- positive
- catalase
Staph and the oxidase test?
- contain cytochrome oxidase
- a respiratory chain component that’s readily tested by oxidase test
How to distinguish staph aureus from other staph?
- coagulase test
- enzyme coagulase binds fibrinogen in plasma and triggers cascade of reactions leading to blood clotting
- most staph aureus produce coagulase but other staph don’t
- aureus also produces one or more DNase enzymes which can help it escape from host DNA released at sites of infection to trap bacteria
Most oral strep are … as they can cause what?
- pathogenic
- infective endocarditis
When is S. salivarius used for good?
- sold in some areas as probiotic
- these strains produce bacteriocins which are peptide antibiotics to help control pathogenic species
Strains of strep produce what? that is used to identify it
Why?
- urease
- enzyme converts urea into basic (high pH) compound ammonia
- helps protect against dental caries
Even though strep can protect against dental caries, how can it contribute?
- S.salivarius can produce acid from sugars
- means levels of this bacteria can be elevated at site of carious lesions
Strep are gram …, non-motile/motile, what shape?
- postive
- non-motile
- spherical/cocci
Most oral strep produce … on blood agar culture. Why?
- green tinge
- hydrogen peroxide’s effect on iron in RBCs
How to differentiate staph and strep?
- strep form chains
- staph form bunches
Hydrogen peroxide is produced by what bacteria? and isn’t degraded. Why?
- strep
- lacks catalase
If you can’t see the shapes of staph and strep, how to differentiate?
- strep aren’t degraded as don’t have catalase
- staph are catalase-positive
Where are E.Coli found?
- human gut
- animals
Are all E.Coli bad?
- most are harmless
- well established as lab model
- pathogenic strains are just not uncommon
Which strains of E.Coli are harmful?
- 157 version of O-antigen and 7 version of H antigen (flagellar protein)
- cause foodborne outbreaks with diarrhoea and vomiting
- most common cause of UTIs in women
E.Coli is gram…, what shape? and what oxygen conditions?
- negative
- rod-shaped/bacillus
- aerobic
Most strains of E.Coli are motile/non-motile and produce fimbriae/don’t
- motile
- do
E.Coli ferments what? and produces large colonies in what agar?
- lactose
- large red colonies in MacConkey agar
How does E.Coli produce energy?
aerobic respiration
How is E.Coli protected against hydrogen peroxide?
- catalase
How do you carry out urease test?
- label agar plates
- label Eppendorf tubes with urease broth inside to correlate with agar plates
- take a full loopful of bacterial cells from each agar and add to corresponding tube of 0.5 ml of urease broth
- colour change is faster if lots of cells are picked
- add broths to water bath and incubate at 37 degrees at end of practical
Possible results of urease test and why?
- urease broth has medium of 2% urea and phenol red as indicator
- any organism that produces urea should change pH indicator from yellow to pink due to production of ammonia
- (NH2)2CO and water becomes carbon dioxide and 2 ammonia
- takes 6-24 hrs to full develop but colour change seen after around an hour
Explain catalase test
- hydrogen peroxide strongly oxidising agent
- using Pasteur pipette, place 3 drops of it on microscope slide and do for and label each bacteria
- take a loopful of cells from each and add to corresponding slide
- dip loop in 6% hydrogen peroxide solutionE
Explain possible results of catalase test
- if bacteria is catalase-positive, bubbles develop in liquid when loop dipped in hydrogen peroxide
- detects catalase enzyme
- 2H2O2 becomes 2 water and oxygen
- bubbles seen are oxygen liberated by catalase
Explain DNase test
- use tripartite plate with 3 species
- using Pasteur pipette, flood each section with Toluidine Blue
- gently rock plate to spread solution evenly
- after a few mins, check for lighter pinkish colour around each bacterial colony
Explain results of DNase test
- DNase test plate contains high molecular weight DNA incorporated into agar
- DNase enzymes produced during bacterial growth degrade DNA around colonies
- toluidine blue turns pink in the presence of free nucleotides or oligonucleotides released by degradation of DNA
Under a microscope, Gram Neg bacteria appear … and positive appear …
- pink
- dark purple
Explain coagulase test
- commercial Staph Latex Agglutination kit and one plate with two species of bacteria as pos and neg control
- resuspend latex reagent by inverting a few times
- dispense 1 drop of latex reagent into each well of test card
- with a sterile toothpick, transfer 2 colonies of each bacteria into respective well and mix
- gently rock to allow mixture to flow over whole test area
- watch for agglutination for 20 seconds
Explain results of coagulase test
- detects for presence of coagulase which is a clumping factor
- in our case, the blue polystyrene latex particles are coated with human fibrinogen
- bacterial cells with clumping factor will bind to fibrinogen and cause them to agglutinate
Results of urease test for
- staph aureus
- E.Coli
- strep salivarius
- neg
- neg
- pos
Results of catalase test for
- staph aureus
- E.Coli
- strep salivarius
- pos
- neg
- neg
Results of DNase test for
- staph aureus
- E.Coli
- strep salivarius
- pos
- neg
- neg
Results of Gram stain test for
- staph aureus
- E.Coli
- strep salivarius
- blue cocci clusters
- red rods
- blue cocci (more highly branched than staph though)
Results of coagulase test for
- staph aureus
- E.Coli
- strep salivarius
- pos
- no test
- no test
Why in the coagulase test did we not test E.Coli or strep?
- because we had confirmed them already
- just needed to confirm staph
Brilliance E.Coli/coliform agar
- selects what species?
- selective agent
- indicator
- Gram-neg bacteria
- sodium lauryl sulfate
- rose-Gal/X-Glu
Schaedler Anaerobe agar
- selects what species?
- selective agent
- indicator
- fastidious anaerobes
- reducing agents cysteine and hydrochloride/glucose
- none
Pseudomonas C-N Selective Agar
- selects what species?
- selective agent
- indicator
- pseudomonas
- cetrimide
- cetrimide
Brilliance Candida agar
- selects what species?
- selective agent
- indicator
- candida
- cloramphenicol
- enzyme substrate
XLD agar
- selects what species?
- selective agent
- indicator
- enterobacteria
- deoxycholate
- xylose, lactose, lysine, thiosulfate
Brilliance Salmonella agar
- selects what species?
- selective agent
- indicator
- salmonella
- novobiocin, cefsulodin
- enzyme substrates
What feature of the cell retains a crystal violet stain?
peptidoglycan
What is the virulence factor in staph?
enterotoxin
What can be seen under a microscope to identify bacteria?
- shape
- size
- capsules
- colour of stain
- structure
- gram neg/pos
Role of fimbriae
- hair like filamentous protein
- sticks cells and aids biofilms
What kind of bacteria grow on a blood agar?
complex
What is the criteria for a good bacteria test?
- specific
- clear to read
- sensitive to low numbers of bacteria
- fast
- easy
- cheap
- reproducible
