Lab Test

Why would it be hard to focus the microscope in the hanging drop procedure?

1. Bacteria are harder to see because a reflection of light in an unstained prep. 2. Immersion oil cannot be used because it causes the coverslip to move 3. The thicker special slide in the cover slip with grease makes it difficult to focus with the change in the working distance

Describe the reactions and the processes involved to produce the reactions in the following in a TSI tube. 1. A/A gas 2. Alk/A H2S 3. Alk/Alk

1. Glucose and/or sucrose, and/or lactose is fermented producing a yellow slant and a yellow butt. Gas produced causes bubbles, splitting off the agar and/or displacement of agar in the tube. 2. Glucose only is fermented producing a red slant and a yellow butt. H2S production is indicated by a black colored precipitate that spreads throughout the butt. 3. None of the sugars are fermented. No change in color or pH and/or redslant and red butt.

What are the 4 main reasons for learning about the normal microbiota? (BONUS)

1. To gain an understanding of the different bacteria at specific body sites which provide greater insight to possible infections that result from injury. 2. To gain knowledge of the possible source and significance of bacteria isolated from clinical infections. 3. To gain knowledge of consequences of overgrowth of those bacteria normally absent. 4. To be aware of bacteria's play in stimulating the host immune system.

How is simple staining different from gram stain in the sense of purpose for each procedure?

Both contrast between the bacteria and background. Both give info. about cell shape, size, and arrangement. Gram stain differs from simple stain in that gram stain differentiates by ...the bacteria into gram + and gram -

What is Brownian movement and how does it differ from true motility?

Brownian movement results from random motion of water molecules bombarding the bacteria and causing them to move. True motility and self propulsion involves the 3 modes of motility.

Explain the reaction how bacteria start with 202 (superoxide) and end up with water and free oxygen, including products and enzymes used in the reaction. What constitures a positive reaction? (KNOW)

Bubbles of oxygen gas in a positive reaction.

What is the importance of catalase to certain bacteria?

Catalase protects against powerful, toxic, oxidizing agents such as hydrogen peroxide, superoxoy which destroy bacterial cellular constituents.

What is the purpose of the OF procedure? List and explain the three reactions that can occur.

Determination of oxidative and fermentation metabolism of carbohydrates or sugars by gram negative bacilli oxidation. Airtube is yellow and oil tube is gree. Bacteria can only utilize the sugar in the presence of oxygen so the airtube turns yellow due to the acid biproducts of oxygen and the oil tube without oxygen can't utilize the sugar so it remains green. 2. fermentation: both tubes are yellow. Bacteria do not need oxygen to utilize the sugar so both tubes turn yellow by the acid biproducts. 3. Nonutilization: air and oil tubes are green. Bacteria does not utilize the sugars. No pH change in any tube.

What are the key ingredients in TSI media that produce the various reactions? Why is one sugar lower in % concentration than the other sugars?

Dextrose, lactose, sucrose, sodium thiosulfate, ferric ammonia citrate. To detect bacteria that ferments, only dextrose. The concentration is 1/10 the concentration of other sugars, small acid produced in the slant oxidized quickly to revert back to alkaline pH or red color. Low oxygen tension in the butt of the tube, keeps an acid pH or yellow color. If hydrogen sulfide is produced from thiosulfate it reacts with the ferris citrate to form a black parcipitate.

What are the 3 different modes of motility?

Gliding, forgeller, cork screw, or bending

Why does gram positive organism stain differently than a gram negative one?

Gram + bacteria have more peptiglycan so it retains more crystal violet. Gram - bacteria have less peptiglycan so it loses more crystal violet. Gram + bacteria have pores thru which crystal violet enters and the iodine constricts the pores trapping the crystal violet. Gram - bacteria have bigger pores so stain is lost with decolorization.

Why are some organisms acid-fast and list 3 organisms that are?

High lipid content in cell wall. Microbacterium, Norcadia, Cryptosporidium.

What is the purpose of the 10% KOH procedure?

If a gram stain bacteria from a pure culture is inconclusive, a KOH prep is made by mixing a colony on a slide with KOH for 30 seconds. Gram - bacteria produce a stream and gram + bacteria will not.

What is being looked for in the oxidase reaction? What constitutes a positive reaction?

Indophenol oxidase oxidizes a redox dye in the reagent resulting in a change of color from yellow to dark purple for a positive reaction.

Why use a wet swab for the skin culture?

It allows more bacteria to be picked up off of dry skin.

Describe how to perform the OF procedure. List materials needed. (KNOW)

Materials: 2 OF tubes, needles, stock culture, mineral oil. Using needles innoculate both OF tubes with stock culture by stabbing each tube 4-5 times but not all the way to the bottom. Overlay one tube with mineral oil. Leave both caps loose. Incubate overnight.

Describe how to perform the simple staining procedure. Include equipment, materials, and the steps involved.

Materials: A primary stain, rinse water bottle, slide, stock culture, microscope, rack, loop, bunsen burner. Transfer stock culture to slide, air dry, heat fix, place slide on rack, flood slide with stain for the required time. Rinse slide with water, blot dry and observe under the microscope.

Describe how to perform the tube motility procedure. Include equipment, materials and steps involved.

Materials: stock culture, inoculating needle, motility media. Using the needle, pick up culture and stab down the center of the media but not all the way down. Carefully remove needle straight out without disturbing the stab line. Incubate tube overnight at 37 degrees. Observe for diffused growth of red color area growing away from stab line for a positive test. A negative test has red color only in stab line.

Why are rectal specimens inoculated to MAC agar versus blood agar?

Most interic pathogens are nonfermentive gram negative bacteria. MAC selects for gram negative bacteria and differentiates bacteria that ferment lactose from bacteria that does not where blood agar does not select.

Can Gram positive or Gram negative organisms be acid-fast? Explain.

No, because these bacteria do not contain the high lipid content in the cell wall that is necessary to retain the acid fast stain upon decolorization.

Do most strict anaerobes produce catalase? Why does this explain why they are anaerobes?

No. Most strict anaerobes cannot tolerate oxygen which is a biproduct of the catalase reaction.

What is being looked for in the indole reaction? What constitutes a positive reaction?

Production of indole from tryptophan. Development of a pink color is a positive reaction.

What can cause a false-positive catalase reaction? Why?

Red blood cells from blood agar can be picked up when picking up a colony. Red blood cells have catalase and will give a false positive reaction.

What does gram variable mean?

Some bacteria from a pure culture will have some cells stain gram + and some cells stain gram -

Why should a control tube with no sugars be used?

Some bacteria utilize other substances besides sugars, producing acid biproducts and producing a yellow color or a false positive. A control tube does not contain any sugars.

What is the major difference between the Zieh-Neelsen and Kinyoun acid-fast stain procedure? What is in the Kinyoun carbofuschsin that is not in the Zieh-Neelsen's and what does it do?

The Zieh-Neelsen uses heat to penetrate the cell wall with carbofuschsin. The Kinyoun uses a wetting agent instead of heat to get stain into cell wall. The Carbafuschsin in the Kinyoun procedure, has tergitol 70M as a wetting agent.

What bacteria produces coagulase and how does the enzyme function to help the bacteria survive? What constitutes a positive reaction?

The bacteria that produces coagulase is staphylococcus aureus. It produces a fibrin clot around itself and avoiding the attack of the host defenses. Formation of a plasma clot in the bottom of the tube is a positive test.

What is the purpose of heat fixing?

To fix the specimen to the slide and to kill the bacteria.

What are the problems with using agar specimens versus broth specimens in making a smear.

Too large an incoculum, results in the occurence of large agrigates. Large agrigates of bacteria pile on top of each other therefore making it hard to see morphological and gram stain results. Broth evenly disperse the bacteria.

Describe how to perform the Kinyoun acid-fast staining procedure, step by step, and the purpose of each reagent used. (KNOW)

Transfer stock culture to slide, air carbofusion for 5 mins. Rinse with water and grip decolorizer on slide to rinse stain from the specimen until there is just a tint of color running off. Rinse with water, place on rack, now flood with briliant green for 3 mins. Rinse with water, blot dry, observe under the microscope. The primary stain, carbofusion, is retained by acid fast, waxy complex in the cell wall. The decolorizer removes the carbofusion from non-acid fast bacteria. The counterstain Brilliant green, stains the non-acid fast bacteria greenish-blue.

Why must young cultures be used in Gram staining?

When gram + bacteria become to old they stain gram -

According to the lab manual what 4 microorganisms are commonly isolated from the skin?

staph epi, cornybacterium, staph aureus, strep veridans. yeast, spacillus species, micrococcus species.

What organism is/are most commonly isolated from the throat? Rectal?

throat: cronebacterium, niesseria, staphep, strppneumo, strepviridans, group A strep, staph aureous. rectal: E. coli, citro bacter faundii, enterobacter species, proteus species, pseudomonas