Complete Acid Fast Stain Lab PDF

Preview

Summary

lab...

Description

SARAH GILDENBERG

LONE STAR COLLEGE - MONTGOMERY

BIOL 2420 - SPRING 2020

Microbiology Lab Notebook Report

Acid Fast Stain - Exercise 3-6 March 17, 2020

Introduction The acid-fast stain is a differential stain used to detect cells capable of retaining a primary stain when treated with an acid alcohol. It is an important differential stain used to identify bacteria in the genus Mycobacterium, some of which are pathogens (e.g., M. leprae and M. tuberculosis, causative agents of leprosy and tuberculosis, respectively). Because so few organisms are acid-fast, the acid-fast stain is run only when infection by an acid-fast organism is suspected.

Purpose The purpose of the acid-fast stain is designed primarily to identify members of the genus Mycobacterium. Because you know ahead of time which organisms should give a positive result and which should give a negative result, it is okay to mix them into a single emulsion. Acid-fast stains are useful in identifying acid-fast bacilli (AFB) and in rapid, preliminary, and provisional diagnosis of tuberculosis (with greater than 90% predictive value from sputum samples). It also can be performed on patient samples to track the progress of antibiotic therapy and determine the degree of contagiousness. A prescribed number of microscopic fields are examined and the number of AFB is determined and reported using a standard scoring system.

Materials • • • • • • • • •

Microscope Clean glass microscope slides Squirt bottle with water Bibulous paper Staining tray Staining screen Slide holder Staining solution Recommended organisms

ID #7330577

1

SARAH GILDENBERG

LONE STAR COLLEGE - MONTGOMERY

BIOL 2420 - SPRING 2020

Procedure Ziehl-Neelsen (ZN) Method: 1. Prepare a slide with up to three emulsions, each in a drop of serum. Use a small inoculum and mix each smear thoroughly to separate the sticky mycobacterial cells, but do not spatter. Air dry and then heat-fix. 2. Perform this step with adequate ventilation. Wearing gloves and chemical eye protection, and the slide on the steaming apparatus, cover the smear(s) with a strip of bibulous paper cut slightly smaller than the slide and apply ZN carbolfuchsin stain. Heat the slide to steaming for 5 minutes. Keep the paper moist with stain and do not boil it. Then remove paper and gently rinse slide. 3. Continue holding the slide with a slide holder. Decolorize with acid-alcohol until the runoff is clear. Use caution when handling the acid-alcohol. Still holding the slide on an angle, gently rinse with distilled water into a staining tray. 4. If not already there, return to your lab station carrying the slide in a Petri dish. Then, place the slide on the staining tray and counterstain the smear(s) with methylene blue for 1 minute. Then gently rinse the slide with distilled water into the staining tray. 5. Gently blot dry in a tablet of bibulous paper or paper towels. Do not rub. When dry, observe under oil immersion. Kinyoun Method: 1.

Prepare a slide with up to three emulsions, each in a drop of serum. Use a small inoculum and mix each smear thoroughly to separate the sticky mycobacterial cells, but do not spatter. Air dry and then heat-fix.

2. Perform this step with adequate ventilation. Wearing gloves and eye protection, place the slide on a rack over a staining tray and apply Kinyoun carbolfuchsin for 5-10 minutes. DO NOT heat the slide. Be sure the staining tray catches any excess stain. Then gently rinse slide with distilled water. 3. Continue holding the slide with a slide holder. Decolorize with acid-alcohol until the runoff is clear. Use caution when handling the acid-alcohol. Then gently rinse slide with distilled water. 4. Return the slide to the staining tray and counterstain the smear(s) with methylene blue or brilliant green stain for 1 minute. Be sure the staining tray catches any excess stain. Then gently rinse slide with distilled water. 5. Gently blot dry in a tablet of bibulous paper or paper towels. (Alternatively, a page from the tablet can be removed and used for blotting.) Do not rub. When dry, observe under oil immersion.

ID #7330577

2

SARAH GILDENBERG

LONE STAR COLLEGE - MONTGOMERY

BIOL 2420 - SPRING 2020

Results

Conclusion Acid fast stain a reddish-purple color and acid fast negative cells stain blue. Notice how most of the Mycobacterium phlei (AF+) cells are in clumps, an unusual state for most rods. They do this because their waxy cell walls make them sticky. Try carefully and gently mixing them a little longer when preparing the slide than you would for other stains. The Staphylococcus epidermidis cells (AF—) are also in clumps, but that is because they grow as grape-like clusters. Each cell's diameter is approximately 1 um. Also notice the characteristic beaded appearance of some AF+ cells (circled).

ID #7330577

3

SARAH GILDENBERG

LONE STAR COLLEGE - MONTGOMERY

BIOL 2420 - SPRING 2020

Follow-Up Questions 1.

How does heating the bacterial smear during a ZN stain promote entry of carbolfuchsin into the acid-fast cell wall? -Heating melts the mycolic acid and allows the stain to penetrate the cell walls. 2. Are acid-fast negative cells stained by carbolfuchsin? If so, how can this be a differential stain? -It uses acid alcohol as a decolorizing agent which extracts the carbolfuchsin from the nonacid-fast cells while ineffective on the acid-fast positive cells. The nonacid-cells are then counterstained with brilliant green to show the difference. 3. Why do you suppose the acid-fast stain is not as widely used as the Gram stain? When is it more useful than the Gram stain? -Acid fastness is a characteristic that is shared by just a few organisms. Many bacterial cells are easily stained with simple stains or using the Gram stain. Acid fast is used when acid fast positive bacteria are suspected.

ID #7330577

4...