Microorganisms

Question 1

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

Answer 1

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

Question 2

Can myobacteria be demonstrated with Gram stain?

Answer 2

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

Question 3

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

Answer 3

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

Question 4

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

Answer 4

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

Question 5

What are the ingredients for Carbol Fuchsin and their purpose?

Answer 5

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.

Question 6

How can using heat help with staining Carbol Fuchsin?

Answer 6

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.

Question 7

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

Answer 7

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.

Question 8

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

Answer 8

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.

Question 9

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

Answer 9

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

Question 10

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

Answer 10

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

Question 11

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

Answer 11

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.

Question 12

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

Answer 12

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.

Question 13

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

Answer 13

Best to use Millipore-filtered water

Question 14

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

Answer 14

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.

Question 15

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

Answer 15

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

Question 16

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

Answer 16

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

M. Leprae is causative organism of leprosy.

Question 17

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

Answer 17

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.

Question 18

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

Answer 18

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

Question 19

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

Answer 19

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.

Question 20

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

Answer 20

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

Question 21

What type of stain is Crystal Violet?

Answer 21

A basic dye to stain G+ bacteria.

Question 22

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

Answer 22

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

Question 23

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

Answer 23

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.

Question 24

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

Answer 24

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

Question 25

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

Answer 25

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.

Question 26

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

Answer 26

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.

Question 27

How does processing affect organisms for staining in histo?

Answer 27

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.

Question 28

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

Answer 28

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

Question 29

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

Answer 29

Use a control that has gram positive and gram-negative bacteria. Great example of a good control is appendix.

Question 30

Why should Crystal Violet be filtered before use?

Answer 30

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

Question 31

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

Answer 31

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.

Question 32

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

Answer 32

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.

Question 33

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

Answer 33

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

Question 34

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

Answer 34

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

Question 35

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

Answer 35

G+ Purple G- Red See slides 14 and 15.

Question 36

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

Answer 36

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

Question 37

What does chromic acid do in Grocott's Methenanine?

Answer 37

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.

Question 38

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

Answer 38

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

Question 39

How do you use the Methenamine Ag solution in Grocotts?

Answer 39

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

Question 40

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

Answer 40

Dark Brown, chocolate colour.

Question 41

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

Answer 41

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.

Question 42

What does gold chloride and sodium thiosulphate do in Grocotts?

Answer 42

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.

Question 43

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

Answer 43

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

Question 44

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

Answer 44

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.

Question 45

What is more stable: Methenamine silver or Ammoniacal silver?

Answer 45

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

Question 46

What happens if the silver is overheated in Grocotts?

Answer 46

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

Question 47

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

Answer 47

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.

Question 48

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

Answer 48

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

Question 49

What colour does Grocott's Methenamine Silver stain things?

Answer 49

Grocott’s Methenamine Silver (slide 19) | • Fungal elements will be black and background green.

Question 50

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

Answer 50

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)

Question 51

What microorganism can Giemsa be used to stain?

Answer 51

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.

Question 52

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

Answer 52

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.