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Histotechnology 2 > Microorganisms > Flashcards

Microorganisms Flashcards

1
Q

What are the main chemicals and their purpose of the Ziehl Neelsen technique?

A
  1. Carbol Fuchsin – demonstrates acid fast bacilli
    Made from: Basic Fuchsin, Phenol (carbolic acid), alcohol
  2. 1% Acid Alcohol – Decolorizer
  3. Methylene Blue – Counterstain
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2
Q

Can myobacteria be demonstrated with Gram stain?

A

Mycobacteria cannot be demonstrated with Gram stain so ZN is needed to be done to see Mycobacteria.

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3
Q

What is the capsule of mycobacteria made of and how would you describe its physical characteristics?

A

Mycobacteria have a capsule that is made up of phospholipids and Mycolic acid. It is a very thick waxy/lipid capsule.

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4
Q

Why is it best to perform Ziehl Neelsen with a staining rack over the sink instead of in a coplin jar?

A

Cross contamination may occur with other slides together at one time in a coplin jar.

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5
Q

What are the ingredients for Carbol Fuchsin and their purpose?

A

Carbol Fuchsin:

  1. Basic Fuchsin - is a basic (positively charged) dye which attaches to the carboxyl groups in the Mycolic acids in the cell wall of the organisms.
  2. Alcohol - solvent for dye
  3. 5% Phenol - accentuator; increases the selectivity and the rate of the reaction.
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6
Q

How can using heat help with staining Carbol Fuchsin?

A
  1. Heat can be added to soften the cell wall to allow for better dye penetration.
  2. It can also help speed up the time it takes for Carbol Fuchsin to stain. Without heat staining takes about an hour, with heat (56 degrees) takes 30 min.

Note: For#2, in lab we did CF for 30 mins, no heat and the textbook seems to suggest with heat it is 15 mins.

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7
Q

What does the 1% Acid Alcohol do in the Ziehl Neelsen technique?

A

1% Acid Alcohol- Decolorizes. It breaks down bonds between the basic dye and the tissue component but cannot break the Basic Fuchsin-mycolic acid complex.

Once Acid-fast bacilli are stained with Carbol Fuchsin they become very resistant to the decolorizer, so decolorizing in this technique is more forgiving.

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8
Q

What method/procedure steps should you take with the acid alcohol and how long is it used on the slide?

A

Flood slides with Acid Alcohol to remove excess Carbol Fuchsin.

To determine the end point of decolorization, the tissue macroscopically should appear a very faint light pink. To stop decolorization from continuing wash slides in tap water well before flooding slides with Methylene Blue.

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9
Q

What type of dye is Methylene Blue in Ziehl Neelsen technique (reminder)?

A

Methylene Blue- a basic dye used to highlight the red acid-fast bacteria.

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10
Q

Why should you only use Formalin fixed tissues with the Ziehl Neelsen technique?

A

Fixation in alcohol fixatives may dissolve the capsule and cause the organism to not be demonstrated.

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11
Q

What is it recommended to do before using Carol Fuchsin? What problems are avoided by doing so?

A
  1. Carbol Fuchsin is a thick dye so filtering before use is recommended.
  2. Filtering Carbol Fuchsin helps to eliminate tons of stain precipitate on slide.
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12
Q

What can happen if you use tap water in your take down before using Carbol Fuchsin in ZN technique?

A

Tap water has noninfectious acid-fast bacilli called M. Gordonae which can give a false positive result on slide. After Carbol Fuchsin step has been completed using tap water is okay.

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13
Q

What is the recommended type of water to use in the ZN technique before Carbol Fuchsin in the take down?

A

Best to use Millipore-filtered water

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14
Q

What problem can overstaining with Methylene Blue cause and how would you remedy it?

A

Overstaining with Methylene blue will mask Mycobacteria. If this occurs need to take back to acid alcohol to remove Methylene Blue and re-stain with Methylene Blue.

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15
Q

What could cause the Methylene Blue counterstain in ZN to stain very light or not at all?

A

If acid alcohol is not rinsed out properly from slide it will cause the counterstain to stain very light or not at all.

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16
Q

What stain technique is used to demonstrate M. Leprae? What disease is associated with M. Leprae?

A

Fites acid-fast stain is used to demonstrate M. Leprae

M. Leprae is causative organism of leprosy.

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17
Q

What technique can be used as an alternative to help better see Acid Fast organisms?

A

Fluorescent technique can be used= Auramine-Rhodamine

Acid-fast bacilli usually are present in small numbers and small to see microscopically so fluorescent technique may be helpful to see organisms better.

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18
Q

Can acid-fast bacilli be seen with an H&E stain?

A

Can’t see Acid-fast bacilli with H&E, special staining technique is needed

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19
Q

What is the result and what can you do if in the ZN technique your slides did not decolourize well?

A
  1. Carbol Fuchsin color will still be on tissue if it did not decolorize well with Acid Alcohol (slide 10). Want to avoid Carbol Fuchsin dye on anything other than the acid-fast bacilli, decolorization needs to be done for longer.
  2. To fix this problem if slides haven’t been taken through DC, flood slides with acid-alcohol longer to remove the pink in the background and then stain with Methylene Blue.
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20
Q

What are the main chemicals and their purpose in the Brown-Hopps Modification of the Gram Stain?

A
  1. Crystal Violet – 1° Stain –> Gram + Bacteria
  2. Iodine – Trapping Agent
  3. Acetone – Decolorizer –> Removes Crystal Violet
  4. Basic Fuchsin - 2° Stain –> Gram – Bacteria and Nuclei
  5. Picric Acid/Acetone – Counterstain –> All other tissue components
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21
Q

What type of stain is Crystal Violet?

A

A basic dye to stain G+ bacteria.

22
Q

How does iodine work as a trapping agent in the gram stain?

A

Iodine- forms a High Molecular Weight Complex (HMWC) in bacteria. Helps to maintain crystal violet in the Gram-Positive bacteria cell wall.

23
Q

Why is acetone used as the decolourizer in the gram stain?

A

Acetone is used as it is a slower process and more easily controlled. The HMWC resists decolorization. If tissue is in acetone too long, then Gram Positive bacteria can decolorize. If tissue is not left long enough in acetone, then gram negative bacteria will look more gram positive as the crystal violet will remain in cell wall.

24
Q

Why is basic fuchsin used as the 2nd stain in the gram stain?

A

Basic Fuchsin- stain Gram negative bacteria and binds more strongly to nuclei through electrostatic attraction than cytoplasm and other tissue components.

25
Q

How does the picric acid/acetone counterstain work on tissues in the Brown-Hopps Modification of the Gram Stain?

A

Picric acid/Acetone- picric acid being a small dye molecule will bind to cytoplasm by electrostatic attraction and stains it yellow. Acetone used a s a differentiator and also begins the dehydration process.

26
Q

What is the final step after picric acid/acetone in the Brown-Hopps Modification of the Gram Stain technique?

A

After Picric Acid/Acetone step there is a final coplin jar for the dehydration and clearing step. The coplin jar is equal parts of Acetone and Xylol. Once the dehydration and clearing step is done, DC in bench top fume hood is no longer needed for this technique.

27
Q

How does processing affect organisms for staining in histo?

A

Microorganisms in tissue have passed through the alcohols and organic solvents of tissue processing. Therefore, some of the lipid components are dissolved out making the gram-negative cell wall more porous than the gram-positive cell wall.

28
Q

Why is formalin fixation preferred for the Brown-Hopps Modification of the Gram Stain technique?

A

Formalin is used because it favors preservation of gram-positive material in cell wall.

29
Q

What type of control should be used for the gram stain technique?

A

Use a control that has gram positive and gram-negative bacteria.

Great example of a good control is appendix.

30
Q

Why should Crystal Violet be filtered before use?

A

Filtering Crystal Violet will help eliminate stain precipitate on tissue/slide.

31
Q

What is the concern with time and acetone in the gram stain technique?

A

Acetone evaporates extremely quickly. At no time should the tissue be allowed to dry out.

Textbook: Drying leads to formation of insoluble compounds that are difficult or impossible to decolorize with picric acid-acetone.

32
Q

Is the staining for Brown-Hopps Modification of the Gram Stain technique done on the staining rack or coplin jars?

A
  1. Staining of microorganisms should be done on slide rack and solutions flooding slides.
  2. The Counterstain portion of technique can be done in coplin jars.
33
Q

How is the grams stain technique be affected if the patient is on antibiotics?

A

Antibiotics compromise cell wall. Gram positive organisms most likely will not stain well if patient is on antibiotics.

34
Q

Why can’t a H&E technique be done instead of a gram stain?

A

Can’t see bacteria with H&E technique. A special staining technique is required.

35
Q

What are the colours of g+ and g- bacteria with Brown-Hopps Modification of the Gram Stain technique?

A

G+ Purple
G- Red

See slides 14 and 15.

36
Q

What are the chemicals and their purpose in Grocott’s Methenamine Silver Impregnation technique?

A
  • 5% Chromic acid – Oxidizing
  • 1% Sodium Bisulfite – Bleaching
  • Methenamine Ag – Impregnation
  • 0.1% Gold Chloride – Toning
  • 2% Sodium Thiosulphate – Fixing
  • Light Green – Counterstain
37
Q

What does chromic acid do in Grocott’s Methenanine?

A

Chromic acid- oxidizing to expose aldehyde groups in chitin and hopefully over-oxidize all the other carbohydrates to non-reactive acids. Chromic acid suppresses other carbohydrates and leaving only carbohydrate chitin to stain with silver. This makes it more specific for fungal cell wall.

38
Q

How does the tissue colour change as it goes from chromic acid to sodium bisulfite in Grocott’s?

A

Before Sodium Bisulfite –> tissue is colored LIGHT ORANGE due to chromic acid, color needs to be removed by bleaching color away.

39
Q

How do you use the Methenamine Ag solution in Grocotts?

A

Methenamine Ag- solution is heated to accelerate reaction (60 degrees). Can use a microwave or a waterbath.

40
Q

What colour is it desired to have the fungus after impregnation of silver is complete in the Methenamine Ag solution?

A

Dark Brown, chocolate colour.

41
Q

What are the ingredients and their purpose to make Methenamine Ag solution for Grocotts?

A

Methenamine Ag solution:

  1. Stock Methenamine - Silver solution (25 mL), provides Ag.
  2. Distilled water (25 mL) - solvent
  3. 5% Borax (2 mL) - maintains alkalinity.

The stock solution is made from:
1. 5% silver nitrate (45 mL)
2. 3% methanamine (900mL)
Note: A white precipitate forms but immediately dissolves upon shaking. The clear solution remains stable for months in a clean amber bottle in the fridge.

42
Q

What does gold chloride and sodium thiosulphate do in Grocotts?

A

Gold chloride- improves contrast and makes reaction product more stable

Sodium Thiosulphate- will stop all chemical reactions from continuing and remove unreacted background silver and gold ions.

43
Q

By what principle does the light green counterstain in Grocott’s stain the tissue background?

A

Light Green- acid dye that is green in color. Binds electrostatic attraction to the cytoplasm and CT and by physical means to nuclei.

44
Q

What type of control should be used for the Grocott’s technique?

A

When staining for fungus can use a positive fungus control, when staining for pneumocystis need to use a positive pneumocystis control.

Make sure you use right control for what you are testing for.

45
Q

What is more stable: Methenamine silver or Ammoniacal silver?

A

Methenamine silver can be made ahead of time and stored in the fridge for a few weeks. More stable than Ammoniacal Silver.

46
Q

What happens if the silver is overheated in Grocotts?

A

Silver should not be overheated as it will lead to non-specific staining.

47
Q

What can happen to your clothing if you are not careful with Grocotts?

A

Can turn hands, clothing, etc black

Beware if you have a hole in your gloves. If the liquid gets in there it will turn you hands black, so replace your gloves if you have a hole.

48
Q

Can fungus be seen with other staining techniques? Which techniques and what colours do they show the fungus in?

A

Fungus can be seen with H&E (blue),
PAS (magenta pink),
Gram stain (purple),
ZN (Variable red).

49
Q

What colour does Grocott’s Methenamine Silver stain things?

A

Grocott’s Methenamine Silver (slide 19)

• Fungal elements will be black and background green.

50
Q

What is the goal when using Grocott’s to stain Pneumocytis spp. and how is this goal achieved?

A

When staining Pneumocystis sp. timing may vary when in the silver. Goal is to get the structures to be stained on the outside with a small black dot in the inside (Bullseye structure)

51
Q

What microorganism can Giemsa be used to stain?

A

Can be used to stain H.Pylori

H. Pylori can cause gastritis and peptic ulcer disease. If not treated can lead to development of gastric carcinoma and lymphoma.

52
Q

What are some other techniques, besides Giemsa, to demonstrate H. Pylori in tissue?

A

Modified Giesma gives great reproducible results and is very easy to perform.

Warthin Starry - replaced Geimsa as an H. pylori stain (HSC).

Immunohistochemical technique is also great to help identify H. Pylori in tissue.

Histotechnology 2 (10 decks)

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