# 4.2: Procedures

$$\newcommand{\vecs}[1]{\overset { \scriptstyle \rightharpoonup} {\mathbf{#1}} }$$

$$\newcommand{\vecd}[1]{\overset{-\!-\!\rightharpoonup}{\vphantom{a}\smash {#1}}}$$

$$\newcommand{\id}{\mathrm{id}}$$ $$\newcommand{\Span}{\mathrm{span}}$$

( \newcommand{\kernel}{\mathrm{null}\,}\) $$\newcommand{\range}{\mathrm{range}\,}$$

$$\newcommand{\RealPart}{\mathrm{Re}}$$ $$\newcommand{\ImaginaryPart}{\mathrm{Im}}$$

$$\newcommand{\Argument}{\mathrm{Arg}}$$ $$\newcommand{\norm}[1]{\| #1 \|}$$

$$\newcommand{\inner}[2]{\langle #1, #2 \rangle}$$

$$\newcommand{\Span}{\mathrm{span}}$$

$$\newcommand{\id}{\mathrm{id}}$$

$$\newcommand{\Span}{\mathrm{span}}$$

$$\newcommand{\kernel}{\mathrm{null}\,}$$

$$\newcommand{\range}{\mathrm{range}\,}$$

$$\newcommand{\RealPart}{\mathrm{Re}}$$

$$\newcommand{\ImaginaryPart}{\mathrm{Im}}$$

$$\newcommand{\Argument}{\mathrm{Arg}}$$

$$\newcommand{\norm}[1]{\| #1 \|}$$

$$\newcommand{\inner}[2]{\langle #1, #2 \rangle}$$

$$\newcommand{\Span}{\mathrm{span}}$$ $$\newcommand{\AA}{\unicode[.8,0]{x212B}}$$

$$\newcommand{\vectorA}[1]{\vec{#1}} % arrow$$

$$\newcommand{\vectorAt}[1]{\vec{\text{#1}}} % arrow$$

$$\newcommand{\vectorB}[1]{\overset { \scriptstyle \rightharpoonup} {\mathbf{#1}} }$$

$$\newcommand{\vectorC}[1]{\textbf{#1}}$$

$$\newcommand{\vectorD}[1]{\overrightarrow{#1}}$$

$$\newcommand{\vectorDt}[1]{\overrightarrow{\text{#1}}}$$

$$\newcommand{\vectE}[1]{\overset{-\!-\!\rightharpoonup}{\vphantom{a}\smash{\mathbf {#1}}}}$$

$$\newcommand{\vecs}[1]{\overset { \scriptstyle \rightharpoonup} {\mathbf{#1}} }$$

$$\newcommand{\vecd}[1]{\overset{-\!-\!\rightharpoonup}{\vphantom{a}\smash {#1}}}$$

## Kinyoun Staining Procedure

1. Prepare a slide with Mycobacterium smegmatis on one side and Micrococcus luteus on the other side. (Alternatively, both bacteria may be mixed into one smear).

• Be sure to break up clumps of M. smegmatis before staining.

2. Air dry and heat fix as usual.

3. Add carbol fuchsin (primary stain): leave on for 5-7 minutes.

4. Rinse with water.

• Note: not all of the primary stain will be removed by water in this step

5. Decolorize with Acid-alcohol: 1-2 quick rinses

6. Rinse with water.

7. Add methylene blue (counterstain) and leave on for 2-3 minutes.

8. Rinse with water, blot dry, and look at beautiful bacteria.

Note

Acid-fast organisms retain the primary stain and will appear bright red: non acid-fast organisms are decolorized with acid-alcohol and pick up the methylene blue counterstain. Epithelial cells that may be present in a clinical sample will also appear blue

## Schaeffer-Fulton Staining Procedure

1. Prepare a smear with Bacillus subtilis or Clostridium sporogenes on one side of the slide, any other bacteria on the other side

2. Air dry and heat fix as usual.

3. Add malachite green (stains spores) and leave on for at least 10 minutes.

4. Rinse briefly with water.

5. Stain cells with safranin (stains vegetative cells) for 1 minute.

6. Rinse with water, blot dry and look at beautiful bacteria

Note

If the bacterial species is an endospore former, you will see pink vegetative cells as well as green oval-shaped endospores: non-spore formers will appear only as pink vegetative cells.

Color pictures of organisms stained with the Ziehl-Neelson procedure and the Schaeffer-Fulton procedure can be found on the Blackboard Microbiology Review site.

Additional Gram staining: if time allows, your instructor may have you prepare some additional slides with the Gram stain procedure.

This page titled 4.2: Procedures is shared under a CC BY-NC-SA license and was authored, remixed, and/or curated by Joan Petersen & Susan McLaughlin.