Lab work Flashcards
MacConkey’s medium
=selective for gram -ve bacteria
–>due to inclusion of crystal violet & bile salts that inhibit growth of gram +ve bacteria
Selective Medium
=contains compounds allowing growth of organisms of interest while inhibiting the growth of other organisms
Differential Medium
=allows discrimination between two or more organisms by evoking different colours etc.
T.b. rhodesiense
(acute) E & C Africa
Treatment:
Stage of disease
1 = suramin
2 = melarsoprol
T.b gambiense
(95% of sleeping sickness infections due to this sub-species)
W &C Africa
Treatment:
Stage of disease
1= pentamidine
2= eflornithine or nifurtimox and eflornithine combination therapy (NECT)
Why is isolating single colonies on a streak plate culture important
- single colonies allow you to isolate organisms in pure culture and check for contaminants based on colony characteristics
- the inoculum is streaked several times across the plate, each time spreading out the cells
- flaming loop inbetween each streak reduces cells
What procedure would help you discriminate between E. coli and P. mirabilis?
-plate bacteria onto MacConkeys medium.
E.coli ferments the lactose in the medium, producing acid, turning the phenol red indicator pink, while P.mirabilis is a lactose non-fermenter and no colour change is observed
Tests to discriminate between two forms of sleeping sickness
- Blood sample and perform a LATEX/T.b gambiense aggulation assay - if aggulation is observed, patient is infected with T.b gambiense
- No serological test for T.b rhodesiense, so PCR test for presence of the SRA gene (present in rhodesiense but not in gambiense) could be performed
What are bacteriophage?
viruses that infect bacteria
-most are DNA viruses
Replication of filamentous bacteriophage
-eg M13 do not lyse their host cells but are released via budding process
Virulent bacteriophage
eg T4 , lyses and kills bacterial host as it replicates
Temperate bacteriophage
-eg lambda, can undergo a lytic lifecycle or a lysogenic lifecycle where it is replicated in concert with the host genome and may integrate into genome
How does a virus plaque form?
- plaques are formed by lytic viruses
- when the virus infects the host cell, it replicates & lyses the host cell, and then infects the neighbouring cells, in turn lysing them & spreading
- viruses are titred as plaque forming units/ml
What temp do water and soil microbes grow at?
20-25 degrees C
What temp do bacteria of faecal origin grow at?
37 degrees C
Commensal organisms
- live in body without causing harm to host
- eg E.coli
Opportunistic pathogens
-can cause disease if immune system of host becomes compromised eg Staphylococcus aureus
Primary pathogens
- cause disease in healthy hosts
- eg. Trypanosoma brucei
Cell counts
- viable count= cells viable under conditions
- total count = viable cells and dead cells
What might plating a microbial isolate on Hugh and Leifson medium tell you about its biochemistry?
- hugh and leifson medium contains glucose as a C source & pH indicator (bromo blue) blue–> green –> yellow
- distinguish between exaditive & fermentative metabolism of glucose
What media could you use to isolate Neisseria gonorrhoeae
- chocolate agar is a non-selective medium that enriches for Neisseria
- Thayer - Martin agar incorporates abs to suppress growth of normal flora
Eosin-methylene blue agar:
- inhibits growth of gram +ve bacteria (methylene blue)
- contains lactose & lactose fermenters produce acid
- acid channges eosin from colourless –> red (some acid) or colourless –>black (lots of acid)
- hence differential for lactose fermenters (strong and weak) and non fermenters
How would you discriminate between S.aureus and S.epidermidis in the lab?
- plate on nutrient agar: S.aureus colonies are yellow; S.epidermis colonies = white.
- Coagulase test: s.aureus is coagulase +ve; S.epidermidis is coagulase -ve.
- salt agar: S.aureus ferments mannitol turning medium yellow, S.epidermidis does not
E. coli vs E. aerogenes vs E. faecalis in lab:
E. coli & E. aerogenes = gram -ve rods
Enterococcus faecalis = gram +ve coccus
E.faecalis vs S. aureus in lab:
- s. aureus tends to form clumps of cells
- e. faecalis tends to form chains
-both are gram +ve cocci
Epidemic
=one region or country
eg UK foot and mouth disease 2001
Pandemic
=worldwide
eg Influenza A H1N1
Gram +ve bacterial cell
- higher amount of peptidoglycan
- presence of teichoic acid
Gram -ve bacterial cell
- two membranes (therefore periplasmic space)
- presence of lipopolysaccharide
Why don’t gram -ve cells retain the violet stain>?
- due to thin layer of peptidoglycan
- this allows the insoluble complex of crystal violet &iodine that forms inside the cell in the first stages of gram staining to be readily extracted by the alcohol step
Why is the heat step important during malachite green staining of fixed films of bacteria?
- malachite green staining is for visualising endospores
- tough coat on endospores requires vigorous treatment for stains to penetrate
- without heat step, endospores = colourless against the safranin - counter stained cells
