Group # 3 May 13, 2015

Group members:

Belen, Alexis

Bitera, Christine Danica

Buenaventura, Gelsie Rose

Cantre, Francis Godwin

Castillo, Jamila Anne

Exercise 9

The Staining of Bacterial Cells and Their Structures

Introduction

Staining is a technique used in microscopy to enhance contrast in a microscopic

image. Stains and dyes are frequently used to highlight structures in microbes for

viewing, often with the aid of different microscopes. Stains may be used to define and

examine different types of microbes, various stages of cellular life, and even organelles

within individual cells. Gram stain is a very important differential staining techniques

used in the initial characterization and classification of bacteria in Microbiology. Gram

staining helps to identify bacterial pathogens in specimens and cultures by their Gram

reaction (Gram positive and Gram Negative) and morphology (Cocci/Rod). Endospore

staining demonstrates spore structure in bacteria as well as free spores. Relatively few

species of bacteria produce endospores, so a positive result from endospore staining

methods is an important clue in bacterial identification.

Objectives

1. To learn the technique and acquire skills needed in conducting simple, gram

staining and endospore staining.

2. To differentiate bacterial cells on the basis of their Gram stain.

3. To identify and characterize the endospores of bacterial cells.

Materials

Culture plates/slant plates of the following bacteria:

Gram positive: Bacillus subtilis, Micrococcus luteus

Gram negative: Escherichia coli

Glass slides

Small amber bottles with dropper for

reagents

Wooden clothes pin or slide holders

Wash bottles

Inoculating loop

Alcohol lamp

Large plastic cover for table

Masking tape

detergent

Reagents:

Gram staining reagents:

1. Crystal violet (primary stain)

2. Gram iodine (mordant)

3. 95% ethanol (decolorizer)

4. Safranin (counterstain)

Endospore staining reagents:

1. Malachite green (primary stain)

2. Safranin (counterstain)

Procedure

A. Preparation of Bacterial Smear

The clean glass slides were degreased by rubbing a small piece of cotton with

acetone on the slide surface. This allowed the water to adhere and spread well on the

surface of the slide. The slides were then labeled with masking tape. A loopful of

distilled water was placed on the center of the slide, then using a flame sterilized loop, a

colony of the test bacteria was transferred aseptically on the drop of distilled water. A

bacterial smear was prepared by gently mixing the bacteria with water, forming an oval

shaped smear and was air dried to form a translucent film on the slide. The smear was

then heat fixed by passing the slide over an open flame 3 to 5 times.

B. Hucker Method for Gram Staining

The prepared bacterial smears for either Escherichia coli or Micrococcus luteus was

flooded with crystal violet for 1 minute. The dye on the slide was drained off with a wash

bottle into a 500 ml beaker. The slide was then flooded with gram iodine solution for 1

minute then rinsed and drained again. Afterwards, the smear decolorized with 95%

ethanol for 10 to 15 seconds until no more violet stain ran from the slide. The slide was

rinsed immediately with water then was flooded with the counterstain Safranin for 1

minute. Lastly, the slide was rinsed and dried again and left to dry. The prepared slides

were then observed under OIO.

C. Endospore Staining (Schaeffer – Fulton Method)

A prepared smear of Bacillus subtilis was saturated with malachite green and

steamed by holding the glass slide above a small frame for 10 minutes. The dye was

then drained off after 5 minutes and the slide was cooled. The slide was then rinsed

with tap water and counterstained with Safranin for 1 minute. The excess stain was then

drained off after 1 minute and rinsed with tap water and left to dry. The prepared slide

was then observed under OIO.

Data and Results

Escherichia coli (gram staining) Micrococcus luteus (gram staining)

Unknown (gram staining) Bacillus subtilis ( Endospore staining)

Analysis of data

Conclusion

References

Bahrami-Hessari, M., Dedeles, G.R., De Jesus, S.M., Papa, D.M.D.C., Dela Cruz, T.E.,

Quinto, E.A. (2014). Laboratory Manual in General Microbiology (8th ed.). Manila,

Philippines.