LAB 11(Data Sheet 3.7)

Today we will be working out of page 91-94 in your lab manual.

ZIEL-NEELSEN ACID-FAST STAIN

The acid fast stain is a differential stain used to identify only two types of bacteria.

The only organisms that are acid fast (the acid-fast genera) are:

  1. Mycobacterium
  2. Causes Hansen’s Disease (leprosy)
  3. Causes Tuberculosis (TB)
  4. There are also non-pathogenic species
  5. Norcardia (opportunistic pathogens; only cause disease in those with poor immune systems, etc).

The thing that makes organisms acid fast is the wax (mycolic acid) in their cell walls.

Their endospores resist stain because of mycolic acid.

Clinically, it is important to be able to identify these organisms quickly. This test is only used when a patient is suspected of having TB or leprosy. It is especially useful when someone is suspected of having TB; the sample is obtained from the sputum, and an acid-fast stain is performed to give a preliminary diagnosis right away. It can also be performed on patient samples to track the progress of antibiotic therapy and determine the degree of contagiousness. There are 10 million new cases of TB per year, 3 million deaths, and it affects 1/3 of the world’s population.

The results are recorded as AF (acid fast) or NAF (non-acid fast). Don’t record them as positive or negative (like the lab manual says) or you may get them confused with Gram stain results.

In the SF stain technique, the primary stain is applied with heat (steam). The reason for this is that the heat increases solubility of the mycolic acid so it can react with the primary stain. Therefore, heat allows stain to penetrate resistant cells because mycolic acid is waxy. Blotting paper must be used on top of the stain when heat steam is used; it keeps the stain from drying out.

We will be preparing a slide that is a mixture (emulsion) of two organisms:

  1. Mycobacterium smegmatis (AF)
  2. Staphylococcus aureus (NAF)

Mycobacterium smegmatisis not pathogenic (we are only SL-2 here). It is saprobic. That means it lives off dead organic matter. It is part of the normal microbiota of our skin and the oils in our skin. It also likes dirt. So if you don’t wash regularly, this organism will thrive. It especially lives on the external genitalia: under the foreskin of uncircumcised males and the labia majora of females. It produces a cheesy substance called smegma, which has a foul odor. It only takes one day without washing for it to grow. Hospitalized patients nowadays are in such bad shape they can’t take care of themselves very well, so they frequently get smegma. You’ll learn to recognize the smell!

With the ZN technique:

  1. Primary stain is carbolfuscian (lipid soluable; can penetrate waxy cell wall). It will stain both AF and NAF.
  2. Mordant is the steam heat
  3. Decolorizer is ACID alcohol. This will rinse the color out of the NAF only.
  4. Counterstain is methylene blue. This will be taken up by the NAF cells.

Ziel-Neelsen Acid Fast Stain Technique

  1. Get out a hot plate (the kind that has a coil) and set it on high.
  2. Get a metal beaker from your tote box and fill it half way with tap water; place on the hot plate.
  3. Place a wire stain rack on top of the beaker.
  4. Place a wire mesh square on top of the stain rack.
  5. Clean one slide.
  6. Prepare an emulsion:
  7. Place one loopfull of water on the slide.
  8. Remember to grasp the culture tubes by the glass, not the cap!
  9. Add one needle sample of Mycobacterium smegmatus. This organism is very waxy, so you have to tap and mix VERY WELL to break it up completely. Otherwise, it will clump on your slide. Page 92 in your manual shows a slide that is clumpy.
  10. Add one needle sample of Staphylococcus aureus and tap and mix.
  11. Air dry completely to avoid aerosols.
  12. Heat fix.
  13. Cut a SMALL square of bibulous paper that is the size of your smear. Place this square directly on your slide. This keeps the smear from drying out.
  14. When the steam starts showing from the beaker, place the slide on the wire mesh and add ONE DROP of carbolfuscian (the primary stain) to the paper every 30 seconds for five minutes. Too many drops will cause the carbolfuscian to drip into the water beaker and boil. This will release phenol from the stain, which is a dangerous aerosol. After five minutes, turn off hot plate.
  15. When the slide is cool, pick up slide with a clothespin.
  16. Throw out bibulous paper into the regular trash container.
  17. Rinse gently with distilled water.
  18. Decolorize with acid alcohol (in the stain kit). Do NOT use regular alcohol! Apply the acid alcohol with a rock-and-roll agitation for a few seconds until the color rinses clear.
  19. Rinse gently with distilled water to stop the decolorization process.
  20. Take the slide to the sink and counterstain with methylene blue for one minute.
  21. Rinse gently with distilled water.
  22. Blot dry.
  23. Observe under 1000x and oil.
  24. Look for individual cells. You will see AF rods that are purple-pink, and little blue cocci. This mixture is what you would actually see clinically, because a patient will not have a pure culture. Draw pictures on Data Sheet 3.7 and lavel AF rods and NAF cocci.

What does a slide of ZN look like? Check your lab manual.

What does a slide of SF look like? Check your lab manual.