prac exam Flashcards
Why did more bacteria grow on the washed hands side
Washing removed layer of skin lipids, exposing more bacteria to the agar surface
Differentiate between the appearance of staph aureus and epidermidis on mannitol salt agar
Epidermidis does not ferment mannitol, stays pink
Aureus ferments mannitol, turns agar yellow
Three characteristics of staph aureus. How are these characteristics visualised?
Coagulase positive: clot formation in serum
Catalase positive: bubbles with addition of hydrogen peroxide
Gram positive: purple
Why is mannitol salt agar used to select for normal skin flora?
Replicates conditions of the skin: high salinity
Differentiates between pathogenic and nonpathogenic staphylococci: fermentation of mannitol yields yellow colour, indicating presence of pathogenic strains like S. aureus.
What additional information should be obtained before performing a coagulase test on Gram positive cocci?
The coagulase test differentiates S. aureus from other strains of staphylococci. To determine whether the gram positive cocci is staphylococci, a catalase test should be conducted as staphylococci are catalase positive.
List 3 factors that protect the skin from infection:
Many layers of keratinized cells, dryness, high salinity, lipids (sebum) from oil glands
What is coagulase and how does it relate to the pathogenicity of staphylococci?
Coagulase is an enzyme which clots fibrin in blood to form blood clots. This may protect the bacterium from phagocytosis and isolate it from other defenses of the host.
Why is the swab moistened with saline for a skin swab?
So that the bacteria will be transferred from the skin to the swab and onto the agar plate.
What test differentiates the three major species of Staphylococcus?
Coagulase test. Aureus and Intermedius are coagulase positive.
How could S. epidermidis and S. saphrophyticus be distinguished in the Micrococcacae identification key?
They will have different 5 digit keys.
Why are staphylococcal infections frequent among hospital patients?
Weakened immune systems and presence of resistant bacteria like MRSA.
Why is blood agar a differential medium for streptococci?
Allows for differentiation between alpha, beta and gamma haemolytic species.
Is Gram staining of significant importance in the identification of organisms studied in this exercise?
No, only streptococci were used, which are all gram positive.
What enzymatic test would be used to differentiate between staph and strep if the sample is known to be a gram-positive coccus?
Catalase test. Staph is catalase positive while strep is catalase negative.
Which streptococcus is susceptible to optichin?
S. pneumoniae
Which streptococcus is resistant to optichin?
S. pyogenes
Which streptococcus is resistant to bacitracin
S. pneumoniae
Which streptococcus is susceptible to bacitracin?
S. pyogenes
What type of haemolysis is produced by S. pneumoniae?
Alpha- incomplete green
What type of haemolysis is produced by S. pyogenes?
Beta- complete
Name at least 4 types of disease caused by S. pyogenes:
Streptococcal pharyngitis (strep throat), scarlet fever, impetigo, streptococcal toxic shock syndrome, rheumatic fever.
How would you differentiate between alpha and beta haemolysis?
Beta haemolysis is complete haemolysis with a clear zone with a clean edge able to be visualized around the colony while alpha haemolysis is incomplete, producing methemoglobin and a green, cloudy zone around the colony.
Enterobacteriaceae characteristics:
Short, gram negative, non-spore forming bacilli
Which two enterobacteriaceae are pathogenic
salmonella and shigella
Which two enterobacteriaceae are occasionally pathogenic
proteus and klebsiella
Which two enterobacteriaceae are normal flora
e. coli and enterobacter
What does a bracket indicate under the microtube
Fill both microtube and cupule
What does a line indicate under the microtubule
Fill cupule with mineral oil (anaerobic)
How are values allocated in the microtube tests?
For the first test, a positive result receives a value of 1. The second positive test receives 2 and the third positive test receives 4. Negative tests receive 0.
What are the advantages and disadvantages of multi-test systems?
Advantages: minimal storage space, less use of culture media, rapid results, application to a computerized system for microbe identification
Disadvantages: difficulty in obtaining proper inoculum size, media and reagent carryover, using inoculum that is too old, requires knowledge on how to inoculate and interpret results, reader bias, expensive
What Enterobacteriaceae are of medical significance? List and describe the infections caused by 3 of these organisms.
Salmonellae: enteric fevers, typhoid and gastroenteritis. Penetrate the intestinal mucosa to enter blood stream, infecting other organs. LPS endotoxin causes ulceration of the intestinal wall, abdominal pain, nausea, vomiting and diarrhoea.
Shigella: shigellosis (bacilliary dysentery) with ulceration of the large intestine, explosive diarrhoea, fever and dehydration.
E. coli can cause bloody diarrhoea (enterohaemorrhagic E. coli) and are the most common cause of UTIs.
Would similar results be obtained by use of the computer-assisted method and the traditional colour change method? Why?
Yes, because the same reagents/indicator reactions are involved. A species will either be positive or negative, no matter which method is used as long as the reagents and incubation conditions used are the same.
What is the clinical justification for the use of a rapid test procedure such as the Enterotube II system for the identification of enteric microorganisms?
When there are multiple tests needed to identify the bacteria or a need to differentiate between multiple species so that an appropriate treatment can be chosen and commenced in a timely manner.
Which growth media would you choose to differentiate a mixed culture of faecal enterococci, staphylococci and E. coli?
Staphylococci: Vogel Johnson or mannitol salt agar
E. coli and enterococci: MacConkey agar no. 3- E. coli would appear dark red to mahogany, enterococci would grow on this agar but be translucent to light brown in colour.
- Describe how a zone of inhibition is formed.
The disks used contain antibiotics which diffuse out of the disk and into the surrounding media which is inoculated with bacteria. Closest to the disk, the antibiotic concentration is greatest, and bacterial growth will be suppressed. Concentration of the antibiotic decreases with distance from the disk, eventually reaching a concentration that is insufficient to prevent bacterial growth. This results in a ring around the disk where no bacterial growth occurs, surrounded by bacterial growth where the antibiotic concentration is insufficient to prevent growth.
- Based on the size of inhibition, can you determine the antibiotic(s) that is (are) most effective against each organism?
Antibiotics that are the most effective against each organism will have a zone of inhibition of the greatest diameter.
- Some antibiotics can kill or inhibit many different kinds of pathogenic bacteria (i.e. they have a broad spectrum). In this experiment, did you observe an antibiotic that inhibits both organisms?
An antibiotic that inhibits both organisms will have a large, similarly sized zone of inhibition for both species.
- How can the Gram reaction be useful in prescribing antibiotic treatment?
Antibiotics are classified based on their spectrum of action. Some antibiotics will be effective against either gram negative or gram-positive bacteria due to antibiotic resistance properties of these two classes, while some will be effective against both. For this reason, the Gram-status of an organism needs to be known in order to prescribe an antibiotic that will be effective in inhibiting the growth of that organism.
- Based on the density of bacterial suspension, did you find any difference in the size of inhibition zone?
Areas with a high concentration of bacteria will have smaller zones of inhibition compared to areas with a low concentration of bacteria, as a higher concentration of antibiotic would be required to suppress bacterial growth
- Is the disk-diffusion technique measuring bacteriostatic or bactericidal activity? Briefly explain.
Bacteriostatic activity as once the antibiotic concentration is below its minimum inhibitory concentration, the bacteria is still able to grow. If it was bactericidal, no growth would occur even at concentrations of antibiotic below the minimum inhibitory concentration.
- In which growth phase is an organism most sensitive to an antibiotic?
The exponential growth or log phase where bacteria are rapidly multiplying.
- Why is the disk diffusion technique not a perfect indication of how the drug will perform in vivo? What other factors are considered before using the chemotherapeutic agent in vivo?
The agar plate does not involve factors such as body pH, how a drug is metabolized and excreted (interindividual variation in drug metabolism), concentration of bacteria in the body or the possibility of drug resistant strains.
What 3 tests is SIM media used for?
Tryptophan metabolism to indole, hydrogen sulfide production, motility
Describe a positive result in the indole test
Addition of Kovac’s produces cherry red colour
Describe a positive result in the H2S test
Black pigment
Describe a positive result in the motility test
Bacterial growth not limited to stab area
Describe a positive and negative result in the methyl red test
Methyl red turns red, methyl red turns yellow
What two species does the methyl red test distinguish
E coli is positive (red)
E aerogenes is negative (yellow)
What is the purpose of the methyl red test?
Looks at ability to oxidase glucose to acidic end products
What is the purpose of the Voges Proskauer test?
Ability to oxidase glucose to non-acidic end products. Positive result confirms presence of E. aerogenes when used in conjunction with methyl red test.
Which tests are used in conjunction to examine products of glucose oxidation? What media is used?
Methyl red test (acidic end products) w/methyl red, Voges proskauer test (non acidic end products) w/ Barritt’s reagent. MRVP broth.
Describe a positive result in the Voges Proskauer test
Barritt’s reagent forms pink colour
Purpose of citrate test, media used, reagent
Ability to ferment citrate as a sole carbon source, citrate agar, bromothymol blue
Describe positive result for citrate test
Bromothymol blue turns from green to deep blue
Describe the four tests conducted using triple sugar iron agar
Glucose fermentation (bottom)
Lactose/sucrose fermentation (top slant)
SH2 production
Gas production
Describe positive results for the tests conducted using triple sugar iron agar
Phenol red turns yellow if sugar is fermented
SH2 turns medium black
Gas produces air pockets in media
Describe the purpose of the urease test
Ability to degrade urea to ammonia
What species can the urease test distinguish between
Proteus vulgaris (positive) and other lactose non-fermenters
Describe a positive result for the urease test, what organism is indicated?
Presence of ammonia turns phenol red pink/red, proteus vulgaris
Purpose of oxidase test (on filter paper) and positive/negative result
Presence of oxidase enzyme, deep blue to purple vs yellow
Purpose of sabouraud dextrose agar
Selective for unicellular fungi (yeasts) like candida albicans and saccharomyces sevisiae
Positive result for sabouraud dextrose agar
Yeasts produce white creamy colonies
Purpose of MacConkey agar no.3
Selective and differential between enteric gram negative rods
Interpretation of MacConkey agar no. 3
E. coli produces dark red to mahogany coloured colonies
Others produce translucent to light brown coloured colonies
Purpose of vogel johnson agar
Selective for coagulase positive, mannitol fermenting strains (pathogenic staph like staph aureus)
Positive result on VJ agar
Phenol red turns yellow as mannitol is fermented
Staph aureus (coagulase positive) grows as black colonies
Purpose of mannitol salt agar
Selective for Salt tolerant, differentiate between mannitol fermenting organisms (S. aureus-yellow) and non-fermenting
Purpose of clostridium agar and positive result
Selective for Anaerobic gram-positive rods (c. perfringenes), white colony growth
Purpose of pseudomonas agar
Selective for pseudomonas species (gram negative, aerobic, non-fermentative)
Differential for aeruginosa (green to brown colonies)
Purpose of blood agar
Selective for bacteria that produce haemolysin
Differential between alpha, beta and gamma haemolytic
Difference between methyl red and phenol red indicator
Methyl red is red at acidic pH and yellow at alkaline
Phenol red is yellow at acidic pH and red at alkaline
1- Name three essential properties of a typing method.
Reproducibility
Typeability
Discriminatory
Bonus: ease of performance and interpretation
Define reproducibility
ability of the technique to yield the same result when same strain is tested repeatedly
Define typeability
ability to obtain unambiguous positive result for each isolate analysed. Non-typeable isolates are those that give either a null or an uninterpretable result
Define discriminatory
ability to differentiate among unrelated strains. Ideally, each unrelated isolate is detected as unique.
2- Name five desirable properties of a typing method.
Cheap, computerizable, quick, does not use expensive equipment, easy to use
3- Name five phenotypic typing methods
Serotyping (immunological reactions): antigenic determinants expressed by microorganism
Biochemical markers (biotyping): metabolic characteristics of an isolate
Bacteriophage susceptibility (phage typing): susceptibility or resistance to a standard set of phage types
Resistotyping: resistance or susceptibility against set of chemical agents
Bacteriocin typing: Based on the susceptibility to a set of bacterial peptides (bacteriocin) produced by certain bacteria.
Antibiogram typing: Based on the comparison of susceptibility profiles of an isolate to a set of antibiotics.
4- Name five genotypic typing methods
Gel electrophoresis of plasmids (number and sizes)
Restriction endonuclease analysis of chromosomal DNA
Nucleotide sequence analysis
Southern blot analysis of restriction fragment length polymorphisms
PFGE of chromosomal DNA
5- What are the main differences between biotyping and biochemical fingerprinting?
Both are phenotypic. While biotyping looks at a positive or negative result (for example the metabolism of a substrate/ presence of an enzyme), fingerprinting looks at differences between rates of growth or rates of reaction.
What do capsules consist of
Polysaccharides, glycoproteins or polypeptides
What are capsules soluble in
Water
Two steps in capsule staining
- Crystal violet applied to non-heat fixed smear. Cell and capsule take on purple colour.
- Copper sulfate used to wash slide. Acts as a decolourising agent (removes CV from capsule) and as counterstain (stains capsule light blue).
Explain the medical significance of a capsule.
The capsule increases the virulence of bacteria as it protects the bacteria from phagocytosis.
Why must heat fixation be omitted during the preparation of a smear for capsular staining?
Heating of bacteria shrinks the cells, creating a clear zone around the organism that can be mistaken for the capsule.
Explain the function of copper sulphate in this procedure.
Copper sulfate acts as a decolourising agent by removing excess primary stain (crystal violet) from the capsule and as a counterstain by staining the capsule light blue.
Differentiate between spores and the vegetative cell
The spore is a small dormant structure that is produced in and can withstand adverse environmental conditions, as a survival mechanism, whereas the vegetative cell is a normal growing cell that is functional.
3 steps used in spore staining
- Malachite green applied. Spores (because of coat) do not accept stain easily so application of heat is required. Both vegetative cells and spore appear green.
- Spore cannot be decolourised by tap water so remains green. Water removes excess MG from vegetative cell components, which are colourless.
- Safranin is applied as a counterstain, which stains the vegetative cells only.
Why is heat necessary in spore staining?
To improve the permeability of the spore to the primary stain.
Differentiate between the appearance of spores and vegetative cells after staining
Vegetative cells appear red due to safranin while spores remain green from malachite green.
Explain the function of tap water in spore staining.
Removes excess primary stain (malachite green) from the vegetative cell only, as the spore cannot be decolourised by water once stained due to low permeability (it is impervious).
Distinguish between sporogenesis and germination.
Sporogenesis is the process by which spores are produced whereas germination is the process by which the spore develops into a vegetative, fully functional cell.
Distinguishing characteristics of sarcodina (2)
Amoeba (single cell)
Pseudopodia for phagocytosis
Distinguishing characteristics of mastigophora (3)
Flagella for motility
Undergo mitosis
Autotroph and heterotroph
Distinguishing characteristics of ciliophora (3)
Cilia for motility
Heterotroph
Two nuclei: one macronucleus and multiple micronuclei
What is the mode of transmission of Entamoeba histolytica?
Faecal-oral route
Pathogenic mechanism of entamoeba histolytica
The parasite is ingested in cyst form. Trophozoites are released and have cytotoxic effects, invading and penetrating the intestinal mucosa where they adhere to and destroy epithelial cells.
Why would a cyst be advantageous to a parasitic species?
Enables survival in unfavourable conditions eg soil
What role does the invertebrate play in the life cycle of the trypanosomes? Explain.
Act as a vector and intermediate host for the protozoan parasite. The invertebrate eliminate faeces and urine containing the trypanosomes onto human (definitive host) skin near a bite site. The trypanosomes enter the human blood stream.
In malarial infection, the sexually mature parasite is found in which host? Is this true for all other protozoan parasitic infection? Explain.
The sexually mature parasite resides in the salivary glands of female Anopheles mosquitoes, which are definitive hosts for Plasmodium. Yes, this is normally the case.
Differentiate between intermediate and definitive hosts
The definitive host is the one which harbors the adult parasite and where the parasite reproduces sexually. The intermediate host is the host which harbors the larval stage or the asexual forms of the parasite.
3 lactose fermenting enteric bacteria
Most of E. coli
Enterobacter aerogenes
Klebsiella pneumoniae
5 lactose non-fermenting enteric bacteria
Salmonella typhimiurium
Shigella dysenteriae
Proteus vulgaris
Pseudomonas aeruginosa
Alcaligenes faecalis
How does sabouraud dextrose agar select for unicellular fungi (yeasts)
Low pH and high sugar content
How does MacConkey agar no.3 select for enteric bacteria
Contains bile salts which inhibit non-enteric bacteria
REMINDER: also look at dendrograms
Example of a bacteria that forms capsule
Klebsiella pneumoniae
Example of a bacteria that forms spores
Bacillius subtilis (common soil bacteria)
Define trophozoite
Vegetative form of protozoan parasite
Example of sarcodina
Entamoeba histolytica
Example of matigophora
Giardia and trypanosoma gambiense
Causative agent of malaria
plasmodium falciparium
