high concentration, get toxic patient problems, renal failure, so carefully monitor enough
antibiotic to treat test organism but not kill patient.
Susceptibility Testing Methods: Antimicrobial agents impregnated into filter paper disc –
Control on same plate – Control on different plate. Break points; MIC’s; MBC’s.
Antimicrobial Gradient Testing E-test®: Read plates after recommended Incubation.
Read MIC where ellipse intersects scale. E strips are very easy to read. Numbers are
concentration of antibiotic. Very concentration to dilute in a series. Zone of inhibition
smaller with more dilute till intercept 2mg/mL here, which is MIC.
Susceptibility Testing Problems: Organism in tissues – drug penetration. Microbial
biofilms. Mixed cultures. Adverse affect on normal flora. Microbial agent interactions. Selective
toxicity. Too slow! Pure cultures here, doing aerobically, but not in body (is some aerobic in body
tho). Done overnight. Infection of patient, cardiac valve could have biofilm, organism growing
differently.
How do we know a pathogen causes a specific disease? Potential pathogen isolated from or
detected in clinical samples. Recognised syndromes septicaemia, endocarditis, osteomyelitis
meningitis, UTI, pneumonia pharyngitis. Diagnosis / effective treatment of infection not just on
isolating an organism, but in establishing a plausible link between the laboratory findings,
recognised syndromes and the patient's clinical condition-pus cells for ex.
Using the Diagnostic Laboratory: Need quality sample.
Correct Use of the Laboratory: Is your investigation worthwhile? Do you know the information
you want-yes; is the information already available-no; does it affect patient management-yes;
can the lab provide the information-yes. Otherwise, stop and think again. Contact the lab for
information on the best test, type of sample, timing of sample, transport of sample, and
interpretation of results. GIve the lab all relevant clinical information like antibiotic treatment,
recent travel, and special risks like if they sound like they have TB. Positive UTI, no need to
send again nor redo test.
How to associate a microbe with disease? Koch’s Postulates. Adaptations upon Koch’s
postulates. Diagnostic laboratory significance. Epidemiology. Subtypes/strains. Knock out &
complementation. “Omics” based studies: Pathogen discovery, Metagenomics, Proteomics.
Questions: The newspapers have reported a new infectious outbreak of disease. What
approaches could you use to substantiate or refute these claims? What laboratory methods
might support your investigations? Is the Press genuine or not. Epidemiology. Lab to profile and
characterise different types of organisms. 10 SRAs in hospital, might not all be the same.
What is a pathogen [10%] Give examples of different categories of pathogens and mechanisms
that they employ to damage the host [45%]. How can you investigate whether this organism is
responsible for clinical effects [45%]?
Bacteriology Practical One
Meningitis is in CSF. Sample collection first, then growth of microorganism (we do), then further
processing (ID/sensitivity to antibiotics). The REAL sample has been plated on an appropriate
medium and micro-organisms have grown. You have to now decide the significance of the
growth and process it further.
You should be well versed with the different culture media: Blood agar-differential and enriched
media, MacConkey agar-selective and differential, Mannitol salt agar-selective and differential,
, and Nutrient agar. Selective, differential and enriched types. Enriched-nutrient
agar, needs more nutrition. Selective-inhibits some but enriches other.
Blood Agar: General nutrients + 5% sheep (horse now) blood. For fastidious
organisms. Has haemolytic properties. Alpha is dimmer than beta, versus gamma
which is very faint-this is how you differentiate the bacteria.
MacConkey Agar: It contains bile salts and crystal violet which
inhibits the growth of Gram positive organisms. It also contains
lactose which acidifies the media when it is metabolised. Neutral red is the pH
indicator that turns red at pH<6.8 and is colourless at any pH > 6.8. Selective
due to bile salts; differential due to lactose.
Mannitol Salt Agar: MSA contains high levels of salt (~7.5% so
selective) and is commonly used for the isolation of salt tolerant Gram positive
bacteria such as Staphylococci. It also contains mannitol and phenol red pH
indicator for identification of mannitol-fermenting bacteria. Allows for the
differentiation of S. aureus and coagulase negative staphylococci (S.
epidermidis). Differential due to colour differentiation. Coagulase positive (and
some neg) staphylococci are yellow (positive).
You will be processing the culture for identification and sensitivity testing.
You will be deciding which colony will need further processing. Colony is a group
of bacteria derived from one singular bacterium. Two different species on a plate, one bacterium
each, colonies will look different.
Decision of Processing-Sterile: Depends on the details provided and sample type. Any growth
from a sterile site should be processed. Examples of sterile sites are blood culture,
cerebrospinal fluid, and pleural fluid (membrane fluid around the lungs)-never see organisms in
those body fluids. See growth, any growth has to be processed. Anything growing is unusual.
Does not imply that all growth is suggestive of infection.
Blood culture-Staph epidermidis, gram pos cocci. Blood is sterile, this is unusual, but alcohol
wipe cleans skin, needle needs to be cleaned. Hospitalised; IV line-bacteria is a problem.
Non Sterile: Samples from non-sterile sites will grow normal flora. Usually such sites gives rise
to a mixed growth (more than one type of colony). Not all that grows is subject to further
processing. 10 throat swabs, each 5 types of colonies, have to know bacteria commonly
causing infections at those sites. Only bacteria commonly causing infection or a request from
the clinician will guide the decision-diphtheria rare condition, lots of kids vaccinated, clinician
sees membrane on throat, test for diphtheria is ordered. Nonsterile sites are: throat swab, skin
swab, and vaginal swab (fecal swab).
Case Study: A 3 yr old child has high fever and sore throat. On examination, there are pus
spots on the tonsils-tonsilitis, strep throat. A throat swab has been requested for microbiological
analysis for Group A Streptococcus infection; by default they do this as well. 1st day workflow:
Streak throat swab on blood agar (37C, aerobic or anaerobic). 2nd day: Screen plate for Group
A Streptococcus (appear as beta hemolytic-very yellow); process that colony further.
Processing: Gram staining, catalase test, antibiotic susceptibility. Gram pos cocci-catalase.
Gram neg -oxidase. Alpha-sputum-is a different thing, normally in throat but not sputum.
For mixed cultures, we will be providing you some prompts on which organism to look for on
the culture plate. The colony morphology is the guiding factor in this process.
