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Paper Topic:

Gram & Spore Staining

GRAM AND SPORE STAINING

2008

Name

Lab Partner (s ) name

Lab Title : Gram and Spore Staining

Lab number : 14 15

Date : February 27 , 2008

Purpose

The purpose of experiment 14 is to show how important and useful the Gram stain is in distinguishing bacteria according to their cell wall composition . The experiment likewise aims for the student to learn proper Gram staining techniques so that accurate results are achieved

Experiment 15 aims to teach the Schaeffer-Fulton method which is a spore staining technique for identifying bacteria which

produce highly resistant endospores

Materials and methods

Gram staining

Materials

slides with heat-fixed smears

Gram staining kit

wash bottle

bibulous

Method

First , we applied crystal violet to the smears in the slides and let it stand for about 20 seconds , after which we briefly washed the slide with distilled water , allowing the excess to drain off . Then we covered it with Gram 's iodine solution and again , let it stand , this time for a minute before decolorizing

We did the decolorization step by tilting the slide at a 45-degree angle and then washing the slide just until the alcohol that 's running down the slide has become colorless . Next , we again washed the slide with distilled water then covered it with safranin for 1 minute

We finished the staining procedure by washing the slide with distilled water one last time , then blotted it dry with the bibulous before air-drying and finally viewing it under oil immersion

Spore staining : Schaeffer-Fulton Method

Materials

24-36 hour nutrient agar culture of Bacillus megaterium

Electric hot plate

Small beaker

5 solutions of malachite green and safranin

Method

We first prepared a boiling water bath , over which we later steamed (for five minutes ) the slide that we covered with a piece of towel soaked with the malachite green stain . We then removed the slide from the water bath and allowed it to cool , after which we rinsed it with water for 30 seconds

Next , we applied the safranin for 20 seconds , then rinsed the slide again briefly with water to remove the counterstain . We blotted it dry with the bibulous , then examined it with a microscope under oil immersion

Data /Results

Gram staining revealed small purple dots in slide 1 and large purple dots in slide 2 , representing Staphyloccus aureus and Micrococcus luteus , respectively . Unfortunately , Serratia marcescens and Pseudomonas aeruginosa failed to grow in the culture medium so no staining results were obtained for these two species

Below are the tables summarizing the results of the Gram and spore staining experiments

Gram Staining Results

Slide Staining Results Bacterial Species Classification

1 (no growth ) Serratia marcescens ---

small purple circles Staphyloccus aureus Gram-positive

2 (no growth ) Pseudomonas aeruginosa ---

big purple circles Micrococcus luteus Gram-positive Spore Staining Results

Bacterial Species Staining Results Interpretation

Bacillus megaterium Pink oval shapes with green dots in the middle Bacillus megaterium is an endospore-producing bacterium Questions

Section B

Which of the 3 differential stains would likely be the first used when...

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