Microbiology Lab Exam #3
What is a typical temp, pressure, and exposure time for sterilization by autoclaving?
121 degrees C for 15 minutes at 15 psi
Explain how the Kirby-Bauer test works. Why do different antimicrobials have different zone-of-inhibition standards to determine bacterial sensitivity? (see p. 108)
A filter paper disc with an antibiotic on it is placed on a plate of bacteria where it does or does not cause a zone of inhibition that can then be compared to the antibiotic standard chart Some antibiotics are smaller particles and disperse faster through the agar producing a larger zone
Explain the difference between antiseptics and disinfectants
Antiseptics: inhibits or kills microbes , sufficiently nontoxic and can be applied to skin and mucous membranes Disinfectants - rids an area of pathogenic microorganisms, kills veggies but not endospores, sufficiently toxic and should not be applied to body surfaces
Explain the difference between autoclaving, pasteurization, boiling, and Tyndallization. How hot does each get? Which can sterilize? With what materials or in what situations would you use pasteurization, or Tyndallization instead of autoclaving?
Autoclaving is form of moist heat sterilization conventionally performed at 121 degree C for 15 minutes. Used for sterilization. Pasteurization is a moist heat process used to kill pathogenic bacteria and reduce the number of nonpathogenic bacteria, 63 degree C Boiling is moist heat treatment that kills vegetative forms of most pathogens, performed at 100 degrees C. Tyndallization is the process of using repeated cycles of heating and incubation to kill endospore-forming bacteria. Tyndallization can sterilize usually food.
Which are more likely to survive boiling: bacterial endospores or vegetative cells?
Bacterial endospores because an endospore is a dormant, tough structure.
Explain what the three possible hemolysis responses on a blood agar plate look like. What are the bacteria lysing when they produce a clear zone?
Beta hemolytic - clear, lysing the red blood cells Alpha hemolytic - green Gamma hemolytic - no halo
What are some advantages of the membrane-filter technique over the multiple-tube fermentation test?
Better reproducibility of results, greater sensitivity, because larger amounts of water can be used, and shorter time
Describe the role of deoxycholate, lauryl sulfate, and lactose in the m-Endo LES agar we used in the water filtration lab. Why is each of those present in the media, and do they provide selective or differential properties to the media?
Deoxycholate and lauryl sulfate: Who only wanted to see/test for the gram - coliforms so not having gram + bacteria show up/grow is helpful Selective Lactose: we only wanted to test for the presence of lactose fermenting coliforms, so seeing this green tells us it is a lactose fermenting microbe Differential
Why are two different interpretive standards given for Penicillin G in Table 14.1?
Different bacteria react differently to the drug
What is the difference between direct transmission and indirect transmission of a pathogen? Be able to provide examples to illustrate each.
Direct: when individuals have person-to-person contact ex. Transmission of fecal organisms when touching unwashed hands during handshaking, or skin contact with a lesion caused by herpes Indirect: when individuals transfer microbes to another person via respiratory droplets, ex. Coughing or sneezing, or on fomites
Why did the vegetative E. coli cells suffer greater damage than the Bacillus endospores?
Endospores are much harder to kill than vegetative cells so need more time when killing
Why should you not look directly at a UV light?
Extended exposure to the sun's UV rays has been linked to significant eye problems, including cataracts, macular degeneration, pinguecula, pterygia and photokeratitis.
Explain the mechanism of action for sulfa drugs... how do they prevent microbial growth? Why does this drug not affect humans?
Folic acid synthesis, prevent the formation of folic acid that is needed to carry out several different functions Mammalian cells are not susceptible to sulfonamides as they absorb and use preformed folic acid
How would you set up an experiment to determine TDT or TDP?
For TDT. Use 40, 60, 80, and 100 degrees celsius water baths. Heat the bacteria in the water baths for 10 minute segments putting a sample of the broth in sterile broth using an inoculating loop at every 10 minute mark to determine in there is growth after it goes in an incubator for 24 hours.
Why would Gram + bacteria be more susceptible to Penicillin than Gram - ?
Gram - bacteria have an outer membrane of peptidoglycan that is harder to penetrate
How does heat kill a microorganism?
Heat can kill microbes by altering their membranes and denaturing proteins. The thermal death point (TDP) of a microorganism is the lowest temperature at which all microbes are killed in a 10-minute exposure.
Which water sources we tested had the highest levels of coliforms? Lowest?
Highest levels was the wabash river and the lowest was the water fountain and sink in the HU dorms
What are some situations where UV light is not useful for sterilization? Explain why.
If any item is covered by the least bit of plastic then the UV light would not be able to go through.
What things are frequently autoclaved in our lab? What are some things that you should not autoclave?
In a lab it is used to sterilize plastic and glass bottles, plastic caps, medical instruments, applicators, and numerous other items that can withstand high temperatures and pressures. Do not autoclave flammable, reactive corrosive, toxic or radioactive materials, liquids, or oil
If you had β-hemolytic colonies on your blood plate from Exercise 23, did that mean you had strep throat? Why or why not?
It is the bacteria that causes strep throat, but it can be dormant in our bodies, which means that if you have those colonies you do not automatically have strep throat.
Why was Serratia marcescens chosen as the organism to use in the Epidemiology lab?
It will only produce a red pigment when grown at room. It allows us to see who was infected.
Know a bit about each of the 5 cultures we used in Exercise 24 as they relate to human health and the digestive tract (Klebsiella, E. coli, Proteus, Salmonella, & Pseudomonas)
Klebsiella - part of the normal biota of the colon, but some strains cause pneumonia. It causes urinary tract infections E. Coli - a constant part of the intestinal biota of almost all humans. Usually not pathogenic but is an opportunist. Major source of urinary tract infections and causes diarrhea Proteus - non pathogen found in the colon but causes urinary tract infections especially from catheters Salmonella - grows in intestines and causes diarrhea Pseudomonas - a gram - rod found in the normal biota of the colon as well as in soil, can cause serious wound infections especially in burn patients
In Exercise 31 we assumed that every contact between an infected and uninfected person resulted in a transmission of the disease. Are transmission rates typically 100% in real life? Why or why not?
No, microbes in real life must have their ideal qualities to infect a person, such as the right portal of entry. Also, a person who comes in contact with a pathogen may have already built up resistance to it due to previous exposure or could rid their body of the microbe before it can infect i.e. handwashing
Are all sore throats caused by bacteria? (i.e., will antibiotics cure all sore throats?)
No, some could be viruses
What are some situations where UV light can be useful for sterilizing things?
One example is in the food industry where UV lamps are used for irradiation. Ultraviolet light sterilization kills viruses, bacteria, yeast and fungi in seconds, and can also extend the shelf life and nutritional value. UV light sterilization effectively purifies the air without the use of hazardous chemicals.
What are some situations where water needs to be tested for the presence of coliforms? What does a positive (for coliforms) result look like on m-Endo LES agar?
Sewage treatment plant. A positive result would have colonies that have a pink to dark-red center and they may or may not have a golden metallic sheen.
Why did we put out plates in Exercise 12 in a dark incubator?
Some cells potentially killed by UV light recover when exposed to longer wavelengths of light
What are examples of non-pathogenic bacteria that are commonly found in our throats?
Streptococcus Moraxella catarrhalis Corynebacterium Staphylococcus Yeast
Explain why water quality tests examine for indicator organisms (like E. coli) rather than directly for known pathogens.
Testing for pathogens is complicated and time consuming so less suited for routine investigations
In Exercise 31 some of you may have observed non-pigmented (i.e., non-Serratia) colonies on our plates. Where did these colonies come from?
The colonies that were on the plates were from everything else that we touched like the gloves because they are not sterile.
Why does an autoclave pressurize a sample in addition to heating it?
The extra pressure in an autoclave means that water boils at a temperature higher than its normal boiling point—roughly 20°C hotter—so it holds and carries more heat and kills microbes more effectively.
Explain how the membrane filtration technique works. How do you calculate the concentration of coliforms in water based on results from this method?
The filter technique is by placing the filter base into the neck of the erlenmeyer flask then with sterile forceps place a sterile membrane filter on the glass of the filter holder base. Then you uncover the filter funnel and place the lower surface on the top of the membrane filter. Clamp that. Then pour the liquid in wait until it goes through the filter. After that remove the filter paper and placed on the agar plate.
What are biochemical tests used for in a clinical setting?
The purpose of biochemical tests is to identify microorganisms. This is because different microorganisms have specific metabolic profiles.
How does this range of the light spectrum relate to visible light?
The visible light spectrum is the segment of the electromagnetic spectrum that the human eye can view. More simply, this range of wavelengths is called visible light. Typically, the human eye can detect wavelengths from 380 to 700 nanometers.
Enteric bacteria smell pretty bad and some make quite a bit of gas. What does that tell you about, umm... farts?
They are full of bacteria (which is why they smell bad)
Explain why you might observe a few small colonies growing within the zone of inhibition around a disc containing an antibiotic, antiseptic, or disinfectant.
Those cells may be more resistant to the drug (could have acquired this)
In a clinical lab, the Kirby-Bauer test is performed with a more strict protocol that uses controlled inoculum size, cultures that are only 2-5 hrs old, and a consistent incubation period of only a few hours. Why is this?
To achieve a more realistic version of what the drug will do on a live specimen
Why did we pasteurize the milk during the yogurt lab? What would have happened if we did not do that? Did we add the culture before or after pasteurization? Why?
To reduce the number of viable cells in the milk. The yogurt would have tons of bacteria and probably would not be a smart thing to eat After, if added before, many of the bacteria in the culture would die and not be able to grow and ferment the milk to make the yogurt
In a clinical setting, what would be the reason for doing Kirby-Bauer test?
To test what antibiotic will combat the bacterial infection Tests potency of antibiotics and drugs against pathogens
What range of light wavelengths are considered to be UV?
Ultraviolet (UV) is electromagnetic radiation with wavelengths from 10 nm to 400 nm, shorter than that of visible light but longer than X-rays.
Explain how UV light kills microorganisms.
When bacteria or another type of microbe is directly exposed to certain types of UV light, the DNA of the cell is damaged, preventing it from replicating. If a cell cannot reproduce, then the cell cannot cause infection
Does milk from the store contain bacteria? Up to what concentration of cells/mL? Why do we not autoclave milk prior to selling it?
Yes, 20,000 bacterial cells/mL, autoclaving would take much longer and is not necessary since pasteurizing the milk for 15-30 seconds kills off most of the pathogenic bacteria and gives it a shelf life of 2-3 weeks, also autoclaving would probably curdle the milk
Can some disinfectants also be used as antiseptics? How?
Yes, but antiseptics usually contain lower concentrations of the disinfectant
Can the normal, resident bacteria of your colon cause infections? How?
Yes, if they enter the blood or infect the bladder or wound they can cause serious disease especially in immunocompromised patients - called opportunists
Why is it important to correctly diagnose and treat strep throat?
can lead to complications like rheumatic fever and glomerulonephritis if left untreated, prevent antibiotic resistance
How are differential and selective media different?
differential media permits the identification of organisms based on the appearance of their colonies, while selective media permits only certain organisms to grow and that aid in isolating one type of organism in a mixture of organisms
What are some examples of disinfectants and antiseptics?
disinfectants: bleach, alcohols, thymol antiseptics: iodine, hydrogen peroxide, boric acid
Are vegetative cells or endospores more resistant to antiseptics/disinfectants?
endospores
What are some reasons why we ferment foods with microbes?
preservation, creates beneficial nutrients, good for digestive health
What are some examples of fermented foods? What microbe is used to create them?
wine, beer, sauerkraut, kimchi, yogurt - made by yeast
Does yogurt contain live bacteria in it? How do you know?
yes, the store bought yogurt was able to turn our milk into more yogurt
