MFD Practicals Flashcards
what is the method and result of the coagulase test
Latex agglutination kit containing latex particles covered in fibrinogen.
Add bacteria to the beads.
Bacteria with coagulase will bind to fibrinogen on the particles and cause them to clump together.
what is the method and result of the urease test
Indicator containing 2% urea and phenol red indicator as a pH indicator.
(NH2)2CO + H2O → CO2 + 2NH3
Changes the indicator from yellow to pink due to the presence of ammonia.
what is the method and result of the catalase test
Solution added to bacteria containing 6% hydrogen peroxide.
2 H2O2 → 2H2O + O2
test
If catalase is present, bubbles will appear due to presence of oxygen.
what is the method and result of the DNase test
Plate bacteria on plates containing high molecular weight DNA incorporated into the agar.
Plates are stained with Toludine blue.
DNase enzyme produced in bacterial growth will degrade the DNA around the colonies.
Causes toludine blue to go from blue to pink.
Staphylococcus Aureus structure
- Grape like clumps of round cells.
- Gram positive.
- Catalase positive.
- Contain cytochrome oxidase, a respiratory chain component that is readily tested via the oxidase test.
- Tests positive in the coagulase test.
- Produce one or more extracellular DNase enzymes which may help it to escape from host DNA that is released at sites of infection to trap bacteria.
Staphylococcus Aureus habitat
Found in the human body where they habit the nose (anterior nares), throat (nasopharynx) and skin.
Often found in high salt environments hence they can withstand sweat etc
•means that you can culture Staph alone in high salt growth medium as other bacteria cannot grow on this.
Staphylococcus Aureus culture
High salt growth mediums.
Staphylococcus Aureus Pathogenic Nature
An opportunistic pathogen that causes a lot of infections.
- Toxic shock syndrome and food poisoning
- caused by enterotoxins - Impetigo
- Wound infections
- Systemic infections
- infective endocarditis
why is MRSA a problem
Methicillin resistant Staph. aureus (MRSA) are becoming a real problem as many of them are resistant to antibiotics.
what do Most oral streptococci have the potential to do (except one- name it)
cause infective endocarditis (potentially pathogenic)
except Streptococcus salivarius
what are the benefits of Streptococcus salivarius
• S. salivarius however is sold as probiotics. They produce bacteriocins which are peptide antibiotics that help to control pathogenic species.
Structure of Streptococcus salivarius
- Gram positive
- Non-motile
- Spherical (cocci) shaped
- Usually forms strips of streps (this is how they are distinguished from Staphylococcus which form bunches of grapes).
- Catalase negative.
how does s.salivarius protect against caries
Contains urease, an enzyme that converts urea into the basic (high pH) compound ammonia.
• beneficial in the mouth as it protects from caries
how does s.salivarius appear on culture
Form a greenish tinge on blood agar.
• arises from the effects of hydrogen peroxide, which is produced by the Streptococci on the iron in red blood cells
s.salivarius Pathogenic Nature
Produces acid from sugars.
Can be elevated at the sites of carious lesions.
Can cause infective endocarditis.
E.coli structure
- Gram negative
- Rod shaped (bacillus).
- Aerobic
- Motile
- Produce fimbriae.
- Catalase positive
E.coli Culture
Ferments lactose and produces large red colonies on MacConkey agar.
E.coli Pathogenic Nature
Strains possessing the 157 version of the “O” antigen (which is part of LPS) and the 7 version of the “H” antigen (which is a flagellar protein) cause foodborne outbreaks.
• diarrhoea
• vomiting
Can also cause urinary tract infections.
ecoli test results (how can it be distinguished from s.aureus and s.salivarius)
urease negative catalase negative dnase positive gram negative coagulase negative
ONLY one gram negative
S. salivarius test results (how can it be distinguished from s.aureus)
urease positive catalase negative dnase negative gram positive coagulase negative
ALWAYS CAT NEGATIVE
s.aureus test results (how can it be distinguished from s.salivarius)
urease negative catalase positive DNase positive Gram positive coagulase positive
ALWAYS CAT POSITIVE
what is selective agar
designed to allow the growth of selected bacteria whilst inhibiting (most) others.
what is indicator agar
media include chromogenic tests to identify a particular bacteria.
species selected and selective agents on STAPH AGAR
Staphylococci
Selective agents:
-Sodium chloride
species selected and selective agents on McCONKEY AGAR
Enterobacteria
Selective agents:
-Bile salts
species selected and selective agents on CANDIDA AGAR
Candida
Selective agents:
-Low pH, chloramphenicol
how do the selective agents exploit knowledge of natural habitat of staphylococci
Staphylococci live on the skin in sweaty places. They outcompete other human colonisers in the presence of sodium chloride.
how do the selective agents exploit knowledge of natural habitat of enterobacteria
Enterobacteria, as their name suggests, live in the gut. Here, bile salts inhibit other organisms that are not adapted to live in that environment.
how do the selective agents exploit knowledge of natural habitat of candida
Candida spp., as you might expect, can tolerate low pH. In the mouth, they thrive when dental plaque becomes acidic, as in the case of caries. They also live in the vagina, another low-pH environment. Candida spp. are fungi and are not sensitive to most classical antibiotics, which is why chloramphenicol is included as a selective agent.
Non selective growth media
Fastidious Anaerobic Agar (FAA)
Highly nutritious medium which is used for the cultivation of fastidious anaerobic microorganisms.
Usually non-selective unless antibiotics are added.
Selective growth media
FAA with antibiotics (vancomycin and neomycin)
TYCS (Tryptone, Yeast extract, Cysteine agar + Sucrose)
MSB (Mitis-Salivarius agar + Bacitracin (tellurite omitted)
FAA with antibiotics (vancomycin and neomycin)
Highly nutritious medium which is used for the cultivation of fastidious anaerobic microorganisms.
Antibiotics makes it selective.
TYCS (Tryptone, Yeast extract, Cysteine agar + Sucrose)
Can distinguish between Strep.mutans and Strep.sanguinis.
Favours the plating of Strep.mutans and Strep.salivarius over Strep.sanguinis .
MSB (Mitis-Salivarius agar + Bacitracin (tellurite omitted)
Can usually distinguish between from S.mitis and S.salivarius.
But bacitracin has been added which means that it will select for Strep.mutans.
Contains high levels of sucrose which aids in selection and is also used for the formation of polysaccharides.
TYCS and MSB are both indicator and selective media.
what are kiss plates and what bacteria is mainly present
Blood agar.
• Non-selective
• Highly nutritious
• Both obligate and facultative anaerobes can be plated here but best to use anaerobic conditions and obligate cannot thrive when oxygen is present.
Streptoccoci will be plated here but you can’t distinguish between the different species.
kiss plate- gamma haemolysis
no haemolysis- most bacteria are staph
kiss plate- alpha haemolysis
by oral streptococci producing H2020 which bleaches the haemoglobin
kiss plate Green tinge - ɑ-haemolysis
from the inside of the mouth caused by oral Streptococci. They produce H2O2 which bleaches the haemoglobin.
kiss plate- Red tinge - Ɣ-haemolysis
from the outside of the mouth caused by Staphylococcus.
Complete clearance - β-haemolysis
Whole mouth disinfection
Rinsing the mouth with 0.2% chlorhexidine digluconate
•BHYE plate to spread the colonies.
10ul was added to 20ml of diluent.
•10:20,000 or 1:2000
100ul were then plated onto the blood agar.
You then count the colonies and work out how many are present by cfu.
Before CHX = 3500 x 10 x 2000 = 7 x 107 cfu/ml
After CHX = 18 x 10 x 2000 = 3.6 x 105 cfu/ml
You can then work out the percentage reduction and the change in the number of bacteria
Snyder Test
Used to detect bacteria which are both acid-tolerant (aciduric) and acid-producing 5 (acidogenic) in a sample of saliva.
what acids are formed in the snyder test and what colour change does this form
lactic acid, acetic and formic from glucose
causes the colour to go from blue → yellow.
which bacteria is the most acidophillic (shown in the snyder test)
Lactobacillus spp is the bacteria that is most acidophilic.
- Thought to be one of the main causative agents in caries.
- Now thought that the large numbers are due to the low pH in lesions which selects for them
what does dental plaque look like under the microscope
Under the phase contrast microscope, dental plaque is seen to contain many different bacterial cells, including rods, cocci and sometimes motile bacteria. Bacterial cells tend to clump together and are sometimes attached to epithelial cells (artificially coloured in the micrograph below).
- Why does TYCS distinguish between Strep.mutans and Strep.sanguinis?
The medium uses high sucrose content to promote the formation of specific glucans by S.mutans thus forming colonies.
- Why does MSB select for mutans streptococci?
It contains the antibiotic bacitracin to which S.mutans and S.sobrinus are resistant.
- If the loop contains 10ul and the diluent volume is 20ml what is the approximate dilution achieved when the 2 liquids are mixed?
It is roughly a 2,000 fold dilution (1,000ul = 1ml)
- Which oral microorganism is most likely to grow in the Snyder test?
5.0 inhibits nearly all bacteria apart from Lactobacilli.
- Can you assume that each type of colony represents a different species?
Generally, different colonies WILL be different species. But sometimes strains of a species can produce different colonies (not all strains of Staph contain the characteristic gold pigment). Very similar colonies can also be produced by different species.
- Compare the relative numbers of colonies on the non-selective FAA plates. Is it safe to assume that the colonies represent the total bacterial population? Explain your reasoning.
No. FAA is non-selective but won’t culture everything. Obligate anaerobes won’t be present as they won’t be able to respire anaerobically. Some species in the mouth are also impossible to culture.
- How can you explain the appearance of different colony types on TYCS?
Due to different species.
- Describe the appearance of Porphyromonas, Prevotella and Fusobacterium colonies on FAA.
Porphyromonas and prevotella are almost identicial and can’t be distinguished on colony alone. Fusobacterium look noticeably different.
- Porphyromonas, Prevotella and Fusobacterium spp are usually associated with sub-gingival plaque. Why can the microorganisms survive on the surface of the tongue?
The tongue has a papuillary structure which creates protected pockets that harbour anaerobes. There are also relatively large numbers of desquamated epithelial cells and leukocytes, which act as a substrate for proteolytic bacteria.
Chlorhexidine rinsing procedure
These agar plates were incubated aerobically. What would be the effect of incubating them anaerobically on the number of colonies formed?
Anaerobic conditions allow the growth of both faculative and obligate anaerobes and so the cfu would probably increase.
Inactivation of spores, which bacterial spores survive the heat
Some bacteria have highly resistant spores and it is important that all autoclaves are validated for spore inactivation.
Bacillus survives
Staphylococcus carriage
Everyone is a carrier of Staphylococcus epidermis however, only 30-50% of healthy adults will carry Staphyloccocus aureus in the anterior nares(12-25%) which can cause skin infections that are a common problem in hospitals.
- Can you think of a situation where it would be useful to know the number of bacteria present in a sample?
Clinically viable counts are used to determine the level of the infecting organism. E.g. a urinary tract infections where commensal bacteria are only infectious when there are in larger numbers in the host.
- Is the number of colony forming units (cfu) the same as the number of cells in a population? If not, why not?
No as CFU is an estimate of the number of cells in a population by assuming that all cells in the population will grow when cultured on agar and that all cells give rise to distinct colonies that can be counted.
- If you start with a solution containing 100 mg/ml of amoxicillin and from it prepare 5 further solutions by twofold serial dilution what is the concentration in mg/ml of amoxicillin in the final (5th) dilution?
3.125mg/ml (dilution 1 is 50, 2 is 25, 3 is 12.5g etc etc until you get to 3.125.
- Why is the MIC for a particular antibiotic always lower than the MBC?
MIC = concentration required to inhibit growth
MBC= concentration needed to kill cells
It is easier to stop the growth than to actually kill cells, so it always takes at least as much antibiotic to kill cells compared with simply stopping growth.
- A microbiologist simply reports that the presumptive microorganism is either sensitive or resistant to the antibiotics used in the test. But how does he decide what concentration of the antibiotic to prescribe for the patient?
Antibiotics have to reach the MIC concentration. It all depends on the administration route of the antibiotics e.g. systemically, GI absorption etc.
- In antibiotics sensitivity test, what does the diameter of the zone of inhibition tell us?
It provides an indication of the potency of the antibiotic.
- Sometimes there is an inhibition zone around an antibiotic in which a few colonies have grown. What as occurred?
Called a breakout one. Occurs when the bacteria develop partial resistance to the antibiotics while they are incubated on the agar plate. Allows them to grow where the antibiotic is but at a LOW concentration.
