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Microscope Components and Microbiology Techniques, Exams of Nursing

A.T. Still University of Health Sciences (ATSU)Nursing

A comprehensive overview of the key components and functions of a microscope, as well as various microbiology techniques and staining methods. It covers topics such as the iris diaphragm, nosepiece, objective knobs, objective lenses, steps for using a microscope, the purpose of the oil immersion lens, parfocal microscopes, resolution, and the characteristics of different types of bacterial growth patterns. The document also explains the principles and processes behind staining techniques like the gram stain, acid-fast stain, and capsule stain, as well as the purpose and applications of these stains. Additionally, it discusses aseptic transfer techniques, the use of broth cultures, and the differences between pure, mixed, and contaminated cultures. This comprehensive resource would be valuable for students studying microbiology, biology, or related fields, providing them with a solid understanding of microscope operation and essential microbiological methods.

Typology: Exams

2024/2025

Available from 10/26/2024

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2023MICROBIOLOGY LAB EXAM STUDY GUIDE WITH
COMPLETE SOLUTIONS 2024
Microscope- The Condenser - Answer focuses the light ging into the
objective lens
Microscope- The Iris Diaphragm - Answer adjusts the amount of light going
into the objective lens
Microscope- Nosepiece - Answer holds the objective lenses and rotates to
change from one objective to another
Microscope- Coarse Objective Knob - Answer moves the stage larger
distances to help get the object in focus
Microscope- Fine Adjustment Knob - Answer moves the stage smaler
increments to help get the object in better focus
Microscope- Objective Lens - Answer magnifies the object. It is located
near the object.
Microscope- Occular Lens - Answer magnifies the image of the object from
the objective lens. It is located near the eye.
Steps in using the microscope - Answer 1) scanner power
in place 2) place slide of specimen stage in place with
stage clips
3) Using the course adjustment knob, raise the stage all the way up and
then down slowly until you can see image of specimen
4) Readjust with fine adjustment knob
Describe the purpose of the oil immersion lens - Answer Used when oil is
added to the slide. Oil must touch the object and objective lens to that
the light does not refract futher
Parfocal - Answer Parfocal microscopes are able to move from one
objective to the next and still be in focus due to the length of the objective
lenses
Resolution - Answer The ability to distinguish between two objects that
are very close together.
What is resolving power? - Answer The measurement of resolution
What are two ways to increase resolving power? - Answer 1. By decreasing
the wavelength of light (using a blue filter) 2. Increasing the numerical
aperature
What units are used to express resolving power? - Answer nm or
nanometers
What is better, smaller or larger resolving power? - Answer Smaller is
better Light microscopes have a 200nm resolving power
Electron microscopes have a .2nm resolving power
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2023MICROBIOLOGY LAB EXAM STUDY GUIDE WITH

COMPLETE SOLUTIONS 2024

Microscope- The Condenser - Answer focuses the light ging into the objective lens Microscope- The Iris Diaphragm - Answer adjusts the amount of light going into the objective lens Microscope- Nosepiece - Answer holds the objective lenses and rotates to change from one objective to another Microscope- Coarse Objective Knob - Answer moves the stage larger distances to help get the object in focus Microscope- Fine Adjustment Knob - Answer moves the stage smaler increments to help get the object in better focus Microscope- Objective Lens - Answer magnifies the object. It is located near the object. Microscope- Occular Lens - Answer magnifies the image of the object from the objective lens. It is located near the eye. Steps in using the microscope - Answer 1) scanner power in place 2) place slide of specimen stage in place with stage clips

  1. Using the course adjustment knob, raise the stage all the way up and then down slowly until you can see image of specimen
  2. Readjust with fine adjustment knob Describe the purpose of the oil immersion lens - Answer Used when oil is added to the slide. Oil must touch the object and objective lens to that the light does not refract futher Parfocal - Answer Parfocal microscopes are able to move from one objective to the next and still be in focus due to the length of the objective lenses Resolution - Answer The ability to distinguish between two objects that are very close together. What is resolving power? - Answer The measurement of resolution What are two ways to increase resolving power? - Answer 1. By decreasing the wavelength of light (using a blue filter) 2. Increasing the numerical aperature What units are used to express resolving power? - Answer nm or nanometers What is better, smaller or larger resolving power? - Answer Smaller is better Light microscopes have a 200nm resolving power Electron microscopes have a .2nm resolving power

When is it best to use a agar slant? - Answer To subculture bacteria that does not need to be "isolated" and to store the bacteria for several months. When is it best to use an agar deep? - Answer Used to determine motility and oxygen requirements of bacteria. Describe the process of aseptic transfer from broth to agar slant - Answer

  1. Flame your loop
  2. Hold broth at 45 degrees and pull off cap 3) flame lip of tube
  3. obtain loopful of culture
  4. flame lip of tube and replace cap
  5. pull cap flame lip of slant
  6. insert loop and zig-zag up the butt of the slant 8) flame lip of slant and insert cap Describe the process of aseptic transfer from broth to agar plate - Answer
  7. Flame your loop
  8. Hold broth at 45 degrees and pull off cap 3) flame lip of tube
  9. obtain loopful of culture
  10. flame lip of tube and replace cap
  11. open plate and zig-zag loop (without digging) across the surface of the agar 7) replace lid to plate Why is the top of the tube heated? - Answer To force out air and keep contiminants from settling inside. When is a needle used rather that a loop when transferring bacteria? - Answer 1) To pickup specific colonies without accidently touching other colonies of other species nearby.
  12. Also used to break up large chucks of bacteria 3) Used to inoculate agar deeps define and identify the following broth culture growth patterns -Flocculant - Answer clumps of growth throughout define and identify the following broth culture growth patterns -Pellicle - Answer A film of growth at the top of the broth define and identify the following broth culture growth patterns -Sediment - Answer Precipitate that falls to the bottom of the tube define and identify the following broth culture growth patterns -Turbidity- - Answer Growth evenly distributed throughout.

Describe the purpose of the gram stain. - Answer To distinguish between cells that have a Gram Negative and Gram Positive Cell Wall. Describe the process of the gram stain - Answer o Smear bacteria onto slide as normal. Let dry. o Heat fix. Let cool. o Crystal violet - primary dye - for 1 minute. Rinse with water. o Iodine - mordant - for 1 minute. Rinse with water. o Ethanol - decolorizer - for 30 seconds. Rinse with water (to stop decolorizing). o Gram's Safranin - Secondary dye (counterstain) - 1-5 minutes (depending on cultures and strength of dye.) List the primary dye, counterstain, and decolorizer used in this stain - Answer o Primary - Crystal Violet o Counterstain - Safranin o Decolorizer - Ethanol 95% Discuss the purpose of a mordant and list the mordant used in the Gram Stain - Answer Mordant chemically changes dye so that it is not able to leave the cell wall. It is trapped in the peptidoglycan layer. Describe differences in staining as they pertain to the differences in the structure of the cell wall. - Answer o Gram negative - has an outer membrane of phospholipid that surrounds the thin peptidoglycan layer. It is able to be dissolved with alcohol and therefore the dye is removed during the decolorizing step with alcohol. o Gram positive - has a thick peptidoglycan layer and no outer membrane of phospholipids. Therefore, it is not decolorized by alcohol. Identify cells as Gram positive or Gram negative according to the color of their gram stain - Answer o Gram positive - purple o Gram negative - red/pink. Explain why alcohol washing step is critical to the Gram staining process. - Answer If no alcohol is applied, then all the cells will remain purple and no distinction between Gram + and - will be seen. Describe the purpose of the Acid-Fast Stain - Answer o Purpose - to distinguish between cells that are acid-fast and those that are non-acid fast. To determine if bacteria in sputum is acid fast, indicating that it might be Mycobacterium tuberculosis Describe the process of the Acid-Fast Stain - Answer o Smear bacteria on slide as normal (however, with Mycobacterium it is usually clumpy and will require a needle to separate the clumps in the smear.) Air dry. o Heat Fix. Let cool. o Apply Carbol Fuchsin dye to bacteria for 15 minutes over boiling water to steam the dye into the bacteria. Rinse with water. o Decolorize with Acid Alcohol for 30 seconds. Rinse with water.

Identify the following: primary dye, counterstain, and decolorizing agent. - Answer o Primary Dye - Carbol Fuchsin o Counterstain - Methylene Blue o Decolorizer - Acid Alcohol (3% Hydrochloric Acid + 95% Alcohol) Explain why cells that are acid fast appear red and non-acid fast appears blue. - Answer o Acid fast cells will be stained by the carbol fuchsin and will appear red because they do not decolorize easily (not even with acid alcohol.) o Non-Acid Fast cells - will decolorize with acid alcohol and when counterstained will appear blue (from the Methylene blue dye.) o If only alcohol was used for the decolorizer, some non-acid fast cells would not be decolorized (Gram + cells that were non-acid fast would retain the dye because the 95% alcohol is not strong enough to decolorize them. To decolorize non-acid fast- gram positive cells, you must use acid alcohol.) Describe differences in the cell wall of acid-fast and non-acid-fast cells - Answer o Acid fast cell walls have a waxy cell wall - due to the Mycolic acid in their cell wall o Non-acid fast cells do not have the waxy cell wall - no Mycolic acid. Give examples of disease-causing acid-fast bacteria - Answer o Tuberculosis and Leprosy are caused by bacteria in the Mycobacterium genus. Capsule stain Describe the purpose of a capsule stain - Answer to view the capsule or determine if a capsule is present Describe the process of the capsule stain - Answer o Smear as usual. o No Heat Fixing. Let it air dry. o Apply an acidic dye (like Nigrosin or India ink) in a thin smear - let air dry. Remove excess dye. NO RINSE. o Apply a basic dye on the stained slide for 1 minute. Remove excess dye. NO RINSE. o Blot dry. View List two functions of a capsule - Answer o Anti-phagocytic factor - makes it difficult for a phagocyte to engulf and "digest" it. o Keeps the bacteria from drying out. Identify the name and type of dye used to stain the cells in a capsule stain

  • Answer Basic dyes, like Maneval's, stain the cell of the bacteria. Identify the name and type of dye used to stain the background in a capsule stain - Answer Acidic dyes stain the background around the bacteria Describe the appearance of bacteria and capsules when using this stain

technique - Answer Bacteria are surrounded by a halo of white (the capsule) with a dark background

The dissolving temperature is between 97-100 ˚C. The solidifying temperature is 42 ˚C.

Describe the purpose of the streak plate and pour plate - Answer Streak plates allow isolation of colonies of a sample of bacteria on the surface of an agar plate from undiluted samples. The bacteria are diluted from one section of the plate to the next by flaming the loop and only bringing a small portion of bacteria to the next section of the plate (by streaking). Spread plates allow isolation of colonies of a sample of bacteria on the surface of an agar plate from diluted samples. Pour plates allow isolation by dilution of bacteria to levels that allow isolated colonies throughout agar. Compare and contrast these three techniques by pattern of growth on the surface - Answer Streak plates require dilution by flaming the loop after streaking each quadrant and only taking 3 streaks from the previous quadrant into the new quadrant. By doing this, each quadrant is more dilute than the previous quadrant. The 4th quadrant should always be dilute enough to contain isolated colonies. Pour plates have isolated colonies of bacteria evenly spread throughout the medium (not just on the surface, but all throughout the agar.). In the Pour Plate procedure, the bacteria is diluted in sterile "water blanks" prior to plating the bacteria and pouring agar over the bacteria. The bacteria sample that is dilute enough will produce isolated colonies throughout the medium (rather than just on the surface.) Spread Plate procedure is similar, to the pour plate in that the bacteria is diluted prior to inoculation on the plate. The bacteria sample is placed on an agar plate and then spread evenly across the surface of the agar (rather than throughout the medium). Compare and contrast these three techniques by where bacteria grow in the media - Answer Streak Plate - bacteria grow only on the surface Pour Plate - bacteria grow throughout the media Spread Plate - bacteria grow only on the surface Differentiate between a pure culture, mixed culture, and contaminated culture. - Answer Pure - only one type of bacteria is present in a culture. Mixed - two or more types of bacteria are contained within a culture (intentionally.) Contaminated - two or more types of bacteria are found in a culture (unintentionally.) Explain the purpose of inverting plates when incubating. - Answer Inverting plates makes sure that condensed water does not fall on the surface of agar plates. Water that falls on the plate will mix different colonies together and not allow isolated colonies to appear on the surface What is the purpose of the Standard Plate Count? - Answer To determine how many Colony Forming Units/ml are present. What is the range CFUs/plate that are countable? - Answer 30-300 CFUs per plate are statistically significant.

What are two assumptions that are made in the SPC? - Answer 1) That all bacteria will grow in the media and under conditions provided.

  1. That one bacteria results in one colony forming unit. Describe how to make a specific dilution (like 1:10 or 1:100). - Answer To make a 1:10 dilution, 1 ml of the sample is added to 9 ml of sterile water. This gives a 1:10 dilution because the dilution is calculated by amount added divided by the final volume. (1/1+9 = 1/10) Describe how to calculate the dilution, total dilution, and number of CFU's/ml of the original sample. - Answer Calculate dilution by taking the amount added divided by the final volume (ie. If you added 1 ml to 9 ml, then 1/1+9= 1/10 (1 ml added, 10 ml was the final volume). Calculate total dilution by multiplying the dilutions - for example, if we diluted a sample 4 times - 1ml added to 9ml each time - then the dilution is a 1:10 of a 1:10 of a 1:10 of a 1:10. In mathematical sentences, "of" means multiply. So, 1/10 x 1/10 x 1/10 x 1/10 = 1/10,000. (notice that there are 4 dilutions of 1/10 and the final dilution has 4 zeros. Count the zeros in your problem and in your answer to check yourself.) CFU's/ml - The CFU's are colony forming units. Since we assume that one colony is the result of 1 bacteria cell which originally was inoculated on the plate (not two or more that were very close together), we call them colony forming units. The formula is: CFUs/ml = Total CFUs on the whole plate / amount plated x dilution 1. Determine the amount of CFUs on the whole plate. Many questions will give the amount on a 1/4th or ½ of the plate. That is not the whole plate. To estimate the amount on the whole, multiply by 4 (if 1/4th is given) or 2 (if ½ is given).
  1. Determine the total dilution. One thing to remember - if you plate 0.1ml instead of a full 1.0ml, your total dilution on the plate is 1/10 of your dilution. Unless otherwise stated, assume that 1ml of your diluted sample, was plated. Multiply the total CFUs/plate x the inverse of the total dilution. (The dilution is a fraction. When dividing by a fraction, you are essentially multiplying by the inverse of the fraction. Therefore, CFUs x inverse of dilution is another way to look at it.) Convert Fahrenheit to Celsius - Answer C = 5/9 (F - 32) Convert Celsius to Fahrenheit - Answer F = (9xC)/5 + = How many decimel places, to the right of base - Answer Six places --- .000 000 nm= How many decimel places, to the right of base - Answer Nine places --- .000 000 000 6.02x 10 = moving the decimel over how many spaces? - Answer 23