Microbiology Lab Final

Steps for preparing a simple stain

1. Clean slide 2. Draw circle on bottom of slide 3. Add a drop of water to slide 4. Aseptically transfer bacteria by loop to slide 5. Smear bacteria around the slide 6. Let dry for at least 5 minutes 7. Heat fix passing through flame 8. Stain with methylene blue 9. Wash excess with water 10. Blot slide 11. Dry Slide 12. View with microscope

Steps for an Endospore stain

1. Prepare a smear 2. Place smear on rack over a beaker of boiling water 3. Immediately add malachite green dye to the slide 4. Stain slide over heat 5. Remove slide over heat for 5 minutes 6. Rinse with water 7. Counterstain slide with safranin 30 seconds 8. Wash with water 9. Blot dry/view

Steps for an acid-fast staining

1. Prepare a stain 2. Cover smear with carbolfuchsin 5 minutes 3. Rinse with water 4. Wash with acid-alcohol 1 minute 5. Rinse with water 6. Cover with methylene blue 1 minute 7. Rinse with water 8. Blot dry

Steps for a gram stain

1. Prepare a stain 2. Cover smear with crystal violet 30 seconds 3. Rinse with water 4. Cover smear with Gram's iodine 10 seconds 5. Rinse with water 6. Decolorize with alcohol 7. Rinse with water 8. Cover with safranin 30 seconds 9. Rinse with water 10. Blot dry

An 82-year-old man was hospitalized with pneumonia. He was treated with penicillin and discharged after 7 days when his symptoms subsided. 2 weeks later, the man was readmitted to the hospital for pneumonia. No bacteria or viruses were cultured from a biopsy specimen. Broad-spectrum antibiotic therapy was administered. The patient developed pneumonia again after returning home from an 8-day hospital stay. A bronchoscopy showed separate hyphae in his lung tissue. An investigation of his home showed that he did not keep pets but some sparrows had nested outside the living room window; he was not a gardener or outdoorsman. Aspergillus was cultured from his humidifier. 1. What is your preliminary identification of the pathogen? 2. Why was the diagnosis delayed? 3. Why did his infection recur?

1. The preliminary identification is done on the basis of the presence of the septate hyphae. It causes para nasal sinus infections in humans. Also it ran opportunistic fungus and only affects individuals which are immunocompromised. 2. They were looking for a virus or bacteria in the culture not a fungus. If a fungi culture would have been done the diagnosis would have been found. 3. The infection recurred because Aspergillus was in his home and would continue to get the infection without removing the source.

What ingredient makes mannitol salt selective?

75% of NaCl makes MSA selective

In blue-diaper syndrome, a baby's urine turns blue in its diapers following the administration of oral tryptophan. The baby's serum and urine levels of tryptophan are very low. What is causing the blue pigment to appear?

A baby's normal flora and bacterial enzymes.

Define the term yeast.

A microscopic fungus consisting of single oval cells that reproduce by budding and are capable of converting sugar into alcohol and carbon dioxide.

What is a contaminant?

A polluting or poisonous substance that makes samples not pure.

What is the value of Petri plates in microbiology?

Allows you to view the growth of colonies on a flat surface.

What 3 bacterial shapes did you observe?

Bacillus, coccus, and spiral.

What is phenol (carbolic acid) and what is its usual appearance?

Bacteria with a benzene ring attached to it. It controls/stops microbe growth by acting as a disinfectant.

How can you tell whether or not there is bacterial growth in nutrient broth?

Bacterial growth is usually indicated by turbidity.

Why will old gram-positive cells stain gram-negative?

Because in older cells the cell wall begins to degenerate causing the purple stain to not stay inside the cell.

What is the purpose of flaming the the loop before and after use?

Before to kill unwanted bacteria and other microorganisms that may be living on the loop. After- to keep bacteria from spreading to unwanted areas.

Why must the loop be cool before touching it to a culture? Should you set it down to let it cool? How do you determine when it is cool?

Being too hot can kill the bacteria. No do not sit the loop down. After placing it in the fire count to 30 seconds and it should be the desired temperature.

You have isolated a gram positive coccus from a throat culture that you cannot identify as staphylococci or streptococci. A test for one enzyme can be used to distinguish quickly between these bacteria. What is the enzyme?

Catalase is present in staphylococci but not streptococci

What are the different types of whole colony apperances?

Circular (circle) Irregular (blob) Biconvex (leaf shaped) Filamentous (tree shaped) Rhizoid (root shaped)

What does turbid mean?

Cloudy

What does flocculent mean?

Cotton appearance.

Is blood agar selective or differential? Explain.

Differential. It provides a visual change if certain enzymes or biochemical traits are present in bacterial growth.

Of what advantage to Clostridium is an endospore?

Endospores remain dormant until conditions improve and they can survive harsh environments

What are the different margin types?

Entire (circle) Undulate (blob) Lobate (splat) Filamentous (tree shaped) Curled (multi-rings)

What are bacteria using for nutrients in nutrient agar? What is the purpose of the agar?

Fewer microbes can degrade agar, causing it to remain solid. The purpose of using agar is its used as a solidifying agent.

What are the different types of elevations?

Flat Raised Convex (Rounded) Umbonate (lumpy)

What does pellicle mean?

Floating

What evolutionary advantage would there be to the formation of a pellicle in a liquid medium by a bacterium?

Forms colonies and have a greater chance of survival.

How did the results observed on the mannitol salt and EMB correlate to the gram reaction of the bacteria?

Gram negative grows well on the EMB agar, gram positive grows well on MSA.

Assuming you could stain any cell, would an acid-fast organism be gram-positive or gram-negative?

Gram positive, mycolic acid in the coating of the cell wall makes them impermeable to stains. So it doesn't pick up the blue color very well, and its not decolorized by the agent.

What other methods can be used to determine motility?

Hanging drop slide.

Suppose you gram stained a sample from a pure culture of bacteria and observed a field of red and purple cocci. Adjacent cells were not always the same color. What do you conclude?

I would conclude that the culture is old, and has therefore, lost its ability to resist the decolorizer.

What effect does increased magnification have on the field of vision?

Increased magnification will result in a smaller field of view.

Describe the arrangement of cilia on Paramecium.

It goes all around the body of the cell. Peritrichous.

What advantages does the low-power objective have over the oil immersion objective for viewing fungi?

It helps get a better view of the fungi/algae due to those being larger organisms.

What is the purpose of agar in the medium?

It is a solidifying agent.

Why is it desirable that microscope objectives be parfocal?

It is desirable to have the objects parfocal so that you do not have to refocus the microscope every time you switch lenses.

Clinical specimens suspected of containing Mycobacterium are digested with sodium hydroxide for 30 minutes prior to staining. Why is this technique used? Why isn't this technique used for staining other bacteria?

It is used to remove debris and other contaminating bacterial species but can't be used for staining other bacteria as it would be killed by NaOH. Acid fast bacteria have the cell wall with high levels of impermeability that do not allow it to be killed by NaOH.

What happens to the milk when the suspended (colloidal) proteins are hydrolyzed?

It loses opaqueness

What is the purpose of peptone in the medium?

It provides nutrients like carbon and nitrogen to bacteria.

Why is agar preferable to gelatin as a solidifying agent in culture media?

It remains solid because few microbes can degrade it. Also it liquifies at 100 degrees Celsius and stays in liquid form until 40 degrees Celsius. Once in solid form it will remain that way until 100 degrees Celsius is reached.

What is the disadvantage of litmus milk medium in diagnostic tests?

It's unreliable because it can give a false negative

When is a loop preferable for transferring bacteria? When is a needle preferable?

Loop- when you want to smear on a flat surface. Needle-when you have a bacteria that doesn't need bacteria.

Which medium is differential? MAC, MSA, or EMB?

MAC

How do you know the minimum number of different bacteria present on a plate?

Minimum number of bacteria depends on the number of colonies that grew. Each of the colonies represents growth from a single cell. Very different looking colonies are likely different bacteria.

How do mold spores differ from bacterial endospores?

Mold spores contain DNA that allow the cell to function and reproduce. Whereas bacterial endospores DNA is more or less free in the cells cytoplasm. Also bacterial endospores need more nitrogen than mold spores.

In 1882, after experimenting with staining Mycobacterium, Paul Ehrlich wrote that only alkaline disinfectants would be effective against Mycobacterium. How did he reach this conclusion without testing the disinfectants?

Mycobacterium are acid resistant and doesn't have a break down of the cell wall when exposed to acid alcohol but an alkaline substance can break down the cell wall.

How would an endospore stain of Mycobacterium appear?

Mycobacterium's waxy cell wall would retain the green dye, suggesting that they have endospores when they actually do not

Can iodine be added before the primary stain in a gram stain?

No because the iodine causes the crystal violet to chemically bonding to the peptidoglycan cell walls. Chemically bonding prevents the crystal violet from washing away by helping it adhere.

Decarboxylation of an amino acid results in the evolution of carbon dioxide. Would a gas trap, such as that used in a fermentation test, be an accurate measure of decarboxylation?

No. All the gas would have to be trapped. It would be inaccurate.

Will the isolated colonies always be in the 4th sector on the streak plate?

Not always due to flaming the loop between smears.

What does sediment mean?

On the bottom.

If you gram stain human cells, what would happen?

Primary stain would be removed easily because human cells don't have cell walls.

What advantage does Trypanosoma's shape provide?

Provides mobility. The flagella's long slender shape makes it easier to move.

What is the primary use of slants? of deeps? of broths?

Slants- to provide a solid growth surface. Deeps- to allow anaerobic bacterial growth. Broths- to provide large numbers of bacteria in larger space.

What is a disadvantage of the streak plate technique? of the pour plate technique?

Sometimes bacteria will clump together and look like a colony when they are not.

Why does addition of a bacitracin disk to a blood agar plate make a rapid identification technique?

Streptococcus pyogenes are extremely sensitive to bacitracin.

Name 2 ways in which you can enhance the resolving power?

Switch to a higher aperture lens and decreasing the amount of light being used (less wave lengths).

What controls the amount of light reaching the ocular lens?

The diaphragm and the light intensity adjustments.

In the Dorner endospore stain, a smear covered with carbolfuchsin is steamed, then decolorized with acid alcohol and counterstained with nigrosine. Describe the microscopic appearance after this procedure.

The endospores should be very bright pink, the bacteria light pink, and the background purplish-black.

What would occur if water were accidently used in a place of immersion oil?

The image would be altered/unclear.

Which objective focuses closest to the slide?

The immersion oil objective lens.

Suppose you are viewing a gram stained field of rods and purple cocci through the microscope. What do you conclude?

The sample must be contaminated.

How might the acid fast characteristic of Myobacterium enhance the organism's ability to cause disease?

The waxy coating around the cell wall makes it impermeable to many substances, which includes antibiotics. Therefore the cell is more susceptible to disease since it can't take in antibiotics. These cells aren't digested by phagocytes plus the lipids in the wall resist drying out and stress which makes them able to survive harsh conditions.

How can you tell that the media provided for this exercise were sterile?

There was bacterial growth for the bacteria used in the experiment and no other bacterial growth was noted.

Why are yeast colonies larger than bacterial colonies?

They have extremely thick cell walls because the reproduce by budding yeast cell walls have a nucleus and a larger vacuole.

How can fungi cause respiratory-tract infections?

Through inhalation of the fungi spores which will go on to give the person respiratory tract infections by finding a warm moist environment to reproduce.

Since you can't identify bacteria from a gram stain, why might a physician perform a gram stain on a sample before prescribing an antibiotic?

To identify which type of antibiotics to use. There are antibiotics that are gram specific and there are broad spectrum antibiotics that are effective against both gram negative and positive.

Why is aseptic technique important?

To keep contaminates out of the bacteria of choice.

How would you determine whether a colony was a contaminant on a streak plate? on a pour plate?

Too much growth on the plate by multiple other bacteria besides the one being used.

How could you determine whether the turbidity in your nutrient broth tube was from a mixture of different microbes or from the growth of only one kind of microbe?

Transfer the cell sample to a petri dish with agar and see if different types of colonies grow.

Why are Giardia and Trypanosoma they classified into the same phylum? Which two genera in this exercise are most closely related?

Trypanosoma have one flagellum and Giardia has several flagellums . Trypanosoma and Giardia.

Trypanosoma and Plasmodium are both found in blood. How do they differ in their location relative to red blood cells?

Trypanosoma isn't in the blood cell but it is in the plasma and the plasmodium is in the red blood cell.

What diseases are diagnosed using the acid-fast procedure?

Tuberculosis and leprosy

What prevents the cell from appearing green in the finished endospore stain?

Vegetative cells are easily decolorized so the green dye washes off.

What would happen if the plates were incubated a week longer? a month?

Week longer- the growth would be much larger. A month- you would not be able to tell anything about the growth and possibly have multiple samples.

Is the gram stain of significant importance in the identification of the organisms studied in this exercise?

Yes, because you are able to eliminate certain microbes.

What is the decolorizing agent in acid fast stain?

acid-alcohol

What is the decolorizing agent in the gram stain?

ethanol

Which step can be omitted without affecting determination of the gram reaction?

safranin