Medical Microbiology Lab
Terms in this set (70)
Describe the Bauer-Kirby test.
Measures the sensitivity of bacteria to antibiotics by culturing bacteria on solid growth media surrounding sources of a drug.
Explain the significance of colonies observed growing in the zone of inhibition in a Bauer-Kirby test.
The size of the zone inhibition indicates the degree of sensitivity of bacteria to a drug. A bigger area of bacteria free media surrounding an antibiotic disk means the bacteria are more sensitive to the drug the disk contains.
What is the broth method for determining minimum inhibitory concentration (MIC)?
The aim of broth and agar dilution methods is to determine the lowest concentration of the assayed antimicrobial agent (MIC) that, under defined test conditions, inhibits the visible growth of the bacterium being investigated.
List three factors that can influence the accuracy of the test?
1) Whether anything grows in your control growth tube
2) Whether the tube was contaminated with a different organism than the one you are testing
3) Whether you properly sterilized your inoculating loop
If a McFarland 0.5 standard contains 1x10^8 organisms per milliliter, how many bacteria were added to each ampicillin-containing tube in experiment 13.2?
0.1x10^8/10 ml=1.0x10^6=1x1.0^5=100,000 CFV/tube
When performing a broth dilution test, why is it necessary to include a growth control tube? A sterility control tube?
So that way you have a control to know for sure if the bacteria grow, and to tell if your broth was pre-contaminated or sterile.
How can the minimum bactericidal concentration of an antimicrobial agent be determined from an MIC assay?
It can be determined from broth dilution (MIC) tests by subculturing to agar plates that do not contain the test agent, Bactericidal
Could an organism that is susceptible to an antimicrobial agent in laboratory testing fail to respond to it when that drug is used to treat the patient? Explain?
Yes, If the drug amount is not enough to treat the invading organism in the patient, or if the organism does not react to the drug a lot.
Are antibacterial agents useful in viral infections? Explain?
No, antibacterial agents are specifically used to fight bacterial infections and do not effect a viral infection in any way
Why is it better to use the word susceptible rather than the word sensitive in describing an organism's response to a drug? When speaking of the patient, what does the term drug sensitivity mean?
Susceptible is used to describe the affects an antibiotic has on the growth of an organism. Sensitive is used to describe any reaction a patient may have to the agent.
Describe a mechanism of bacterial resistance to antimicrobial agents, other than resistance gene acquisition.
Includes inheritance with cell wall synthesis, inhibition of protein synthesis interference with nucleic acid synthesis, inhibition of a metabolic pathway and disruption of bacterial membrane structure
If the laboratory isolates S. aureus from five patients on the same day, is it necessary to test the antimicrobial susceptibility of each isolate? Why?
No, since the time and sample variable are the same, results of a test should be similar
What are three actions that could prevent antibiotic resistance?
1) Ask if tests will be done to make sure the right antibiotic is prescribed
2) Take antibiotics exactly as the doctor prescribes. Do not skip doses, completed the course treatment, even when starting to feel better
3) Only take what is prescribed do not share leftover drugs
What is a differential stain? Name two examples of such stains.
Staining process which uses more than one chemical stain. Examples are acid fast stain and gram stain
Is a gram stain an adequate substitute for an acid-fast stain? why?
No, it won't hold the stain due to the (mycolic acid) lipid cell wall
When is it appropriate to ask the laboratory to perform an acid-fast stain?
When an acid-fast organism is suspected
Are saprophytic mycobacteria acid fast?
Does the presence of acid-fast organisms in a clinical specimen always suggest serious clinical disease?
How should the acid-fast stain of a sputum specimen from a patient with suspected pulmonary nocardia infection be performed?
Acid-fast method used, different acid-alcohol just acid to decolorize
Why is it important to know whether or not bacterial cells possess capsules, flagella, or endospores
To know what staining technique to use
How can one destroy endospores?
When dry heat is applied at high temperatures or long periods, by steam heat under pressure (autoclave), or by special sporicidal (Endospore-killing) disinfectants.
Is bacterial sporulation a reproductive process? Explain?
No because the mother cell does not survive and only one spore is made. It is a survival mechanism not a reproductive process
Why is it important to determine the location of the endospore within the bacterial cell?
Because the position of the endospore differs among bacterial species and is useful in identification
Can you relate endospore staining to endospore survival in hospital or other environments?
Endospores are high resistant to environmental influences. Most disinfectants cannot permeate it. Because bacterial endospore walls are not readily permeated by materials in solution, the inner contents of the endospore are not easily stained by ordinary bacterial dyes. The relation is that it is not killed or stained by ordinary methods.
What is a negative stain? In what clinical situation might it be used?
It stains the background of the slide and not the organism, small or stain resistant bacteria, leaves an unstained halo around the capsule of the cell.
Describe a flagella stain and explain the principle of its action.
Make flagella thicker in order to see precipitate, a coating of dye over entire flagella length. Basanaline dyes
Compare the usefulness of a flagella stain with that of a wet mount-stain preparation.
To stain allows the flagella to be longer and seen better as opposed to the hanging drop and saved and looked at later, cannot save wet mount because its alive. wet mount moving and hanging drop you can save
Of what value is a capsule to a microorganism?
Prevent phagocytic cells from ingesting and killing bacteria, protect from desiccation, provide a food reserve when certain organic compounds are in excess, a virulence determinant of pathogenic microbes, and they serve as binding or adhesion agents for sticking cells together.
When an agar plate is inoculated, why is the loop sterilized after the initial inoculum is put on?
So the next streak is not contaminated by the initial inoculum, also because the point is to dilute in a way so by removing all the bacteria from the loop makes it easier to end up with individual colonies
Distinguish between a pure culture and a mixed culture.
Pure culture contains one type of cell
Mixed culture contains many cells
Define a bacterial colony. List four characteristics by which bacterial colonies may be distinguished
Visible bunch of organisms
1) shape of the colony
2) edge of the colony
3) color of the colony
4) Opacity of the colony
Visible to the naked eye
Why does the streaking method you used to inoculate your plates result in isolated colonies?
It "dilutes" the bacteria because after each set of streaks you sterilize your loop and then at the beginning of the next streak you overlap with the end of the one before.
Why was a blood agar, rather than a nutrient agar, plate used for the culture from your mouth?
Because some organisms have complex nutritional requirements that are not easily mimicked with nutrient agar. Using blood allows them to grow better.
Are the large numbers of microorganisms found in the mouth a cause for concern? Explain?
No, the normal flora in the mouth keeps the harmful bacteria from attaching to the mucosa
During their laboratory testing, if disinfectants are carried over into microbial cultures, could the results be affected? Explain?
Yes, it depends on what culture but many disinfectants kill a wide range of microbes. Any chemical added to a culture could change the growth of the culture
Define disinfection. How does it differ from antisepsis?
Disinfection=the destruction of pathogenic microorganisms. A process involving chemical interactions between a toxic antimicrobial substance and enzymes or other constituents of microbial life. A disinfectant must kill pathogens while it is in contact with them so that they cannot grow again when removed. reduces the number of microbes on surface
Antisepsis=reduces the number of microbes on the skin
Define bactericidal, bacteriostatic, virucidal, fungistatic
Bacteriostatic=Inhibits the growth of bacteria
Fungistatic=Inhibits the growth of fungi
Why are control cultures necessary in evaluating disinfectants?
To prove whether or not the disinfectants killed the bacteria, to make sure the bacterial examples weren't contaminated
What factors can influence the activity of a disinfectant?
Exposure time, concentration required to kill microorganisms, temperature, PH, concentration of microorganisms present, and the toxicity of the agent to skin or its effect on the material to be disinfected
Why do microorganisms differ in their response to disinfectants?
Microorganisms of different groups are not uniformly susceptible to chemical disinfection due to the level of resistance of the bacteria
How can bacteriostatic and bactericidal disinfectants be distinguished?
Bacteriostatic= Means that it arrests the growth of bacteria but the bacteria is not killed. It can repopulate the disinfected area once the antimicrobial substance is removed
What is an iodophor? What is its value?
Any group of disinfectants containing iodine in combination with a surfacant. Gradually reduces iodine value is for cleaning and disinfecting equipment and surfaces and as a skin antiseptic
Did you find the mouthwash you tested to be as effective as the other chemical agents included in this exercise? Explain any difference you observed?
No, it did not affect at all after used while the others did. It covered most of the plate not just where the finger tips touched
Why are bacterial endospores a problem in the hospital environment?
They are high resistant, bacterial endospores can cause illness in those with already compromised immune systems in hospitals. Easy for them to cause infection
Briefly discuss disinfection and antisepsis in relation to patient care.
Antisepsis (Chemical disinfection of skin): Hand antisepsis because most hospital aquired infections are transmitted to patients from the hands of the health care workers
Disinfection: Applied to non-living objects to destroy microorganisms that are living there. This is crucial because many patients who have been exposed to contaminated equipment can contract diseases such as HIV
In addition to hand scrub, what other precaution is necessary for health care personnel to prevent spreading infection by their hands when in the operating room?
Make sure to use gloves
Would you expect that long or artificial fingernails interfere with the effectiveness of hand scrubs? Why?
Yes, Because bacteria can hide underneath the nails
Why is it not suitable to wear rings in a hospital setting?
Bacteria can hide in the rings and spread infection
Why should hands be washed before and after dressing changes even though gloves must be worn during the procedure?
To make sure no bacteria are on your hands before you put on gloves, no possible cross contamination from skin to wound. Wash after taking gloves off so you do not cross contaminate
Define differential medium and discuss its purpose?
Differential medium is a culture medium that allows one to distinguish between or among different microorganisms based on a difference in colony appearance (color, shape, growth, pattern) on the medium. Its purpose is to identify certain microbes by seeing how they react with the medium dyes due to the bacterial metabolic properties
Define selective medium and describe its uses?
Selective medium culture medium that allows growth of certain types of organisms, while inhibiting the growth of other organisms. Isolate colonies
Why is MacConkey agar selective as well as differential?
It is selective to gram-negative enteric bacteria because it contains bile salts which inhibit most gram-positive bacteria. It also contains lactose which differentiates between gram-negative lactose fermenter (E.coli) and gram-negative non-fermenters (salmonella)
Why is blood agar useful as a primary isolation medium?
It supports the growth of just about any bacteria. It is nonselective and it is used for primary isolation purposes for aerobic and anaerobic cultures in our lab. It is often used along with chocolate agar and MacConkeys agar as the 1st step in identifying types of bacteria. Almost all can be used on it
How can you distinguish E.coli and P.aeruginosa on MH, Blood agar, and EMB.
MH- E.coli is white and P.aeruginosa is yellow
Blood agar-E.coli is pink with red background and P. aeruginosa is metallic blue or green
EMB- E.coli is Shiny metallic green and P aeruginosa is light pink
What is the major difference between modified thayer-martin (MTM) medium and chocolate agar? when would you use MTM medium rather than chocolate agar?
Chocolate agar is a non-selective medium
Modified thayer- martin is a selective medium for gram-negative bacteria and includes the medium as well as antibiotics. You would use MTM when you are working with a gram-negative bacteria such as Neisseria
If you wanted to isolate S. aureus from a pus specimen containing a mixed flora, what medium would you choose to get results most rapidly? Why?
EMB salt agar, it inhibits organisms like S. aureus due to the presence of methylene blue dye. mannitol salt agar is has high salt concentrations that do not allow other organisms to grow. Only S. aureus can.
What is the value of making a gram stain directly from a clinical specimen?
Gram stain will tell if its a gram-positive bacteria or gram-negative bacteria. This helps to narrow down the pathogen That's associated with the clinical specimen
Why is aseptic technique important in the laboratory? In patient care?
In lab-Contamination may lead to false and inaccurate results
In patient care- bacteria could lead to infections and disease that are resistant to treatment
Why is human blood no longer used in the preparation of culture media?
Because you do not want to be working with something that could potentially kill you. It is a potential health risk for acquiring blood borne pathogens, such as HIV, Hepatitis B, or Hepatitis C
What diagnostic test differentiates proteus and providencia species from other entrobacteriaceae?
Hardy disk urea/PDA dist test
What procedures other than biochemical are used to identify microorganisms?
Morphological characteristics and differential culturing
Describe two mechanisms by which E.coli can produce disease?
By producing toxins and producing structures that enable the microorganisms to attach to the surface at which they cause disease.
What is meant by the term "enteric pathogen"?
Something that affects the GI tract
Name a bacteria pathogen, other than one of the enterobacteriaceae, that causes intestinal disease. Provide a flowchart indicating how you would make a laboratory diagnosis.
1) Label the tubes, inoculate one tube with each medium provided
2) incubate all tubes at 35 degrees Celsius for 24 hours
3) complete the IMVIC and PD tests
Name a rapid method for the identification of enterobacteriaceae, and discuss its value in comparison with the standard methods you have used in exercise 22.
Enterotube 2, Rapid, fast, simple, easy to uses and accurate
Why is it important to differentiate glucose nonfermenters form Enterobacteriaceae?
Because nonfermenters are more highly resistant to common antimicrobial agents. Yellow on the (butt of TSI) indeciates organism is fermentative which is the enterobacteriaceae.
Why must all bacterial identifications be performed with pure cultures?
Because contamination may cause different results. One bacterium can produces its own fingerprint if contaminated may cause a different fingerprint
Which of the microscope objectives is most satisfactory for studying bacteria in wet-mount prepartions?
High-Dry objective because water and oil do not mix so you cannot use the oil-immersion lens
Why must oil be used with the oil-immersion objective?
The oil gives the focal length between the slide and objective lens which controls clarity, resolution, and makes, a column for the light, makes better to see
Clues that help identify an organism; color, shape, desity, gross features
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