Complete Simple Stain Lab PDF

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SARAH GILDENBERG

LONE STAR COLLEGE - MONTGOMERY

BIOL 2420 - SPRING 2020

Microbiology Lab Notebook Report

Simple Stain - Exercise 3-3 January 30, 2020

Introduction Because cytoplasm is transparent, cells usually are stained with a colored dye to make them more visible under the microscope. Then cell morphology, size, and arrangement can be determined. In a medical laboratory, these are usually determined with a Gram stain (Ex. 3-5), but you will be using simple stains as an introduction to these.

Purpose The purpose of this lab is to practice staining and coloring molecules. Staining allows us to tell between gram positive and negative molecules, but this must be done very carefully because if too much is washes away, left for too long it will cause a false positive or negative.

Materials • • • • • • • • • • • •

Microscope Clean glass microscope slides Methylene blue stain Safranin Stain Crystal violet stain Staining tray Squirt bottle with water Staining screen Slide holder Bibulous paper Lens paper Recommended organisms

ID #7330577

1

SARAH GILDENBERG

LONE STAR COLLEGE - MONTGOMERY

BIOL 2420 - SPRING 2020

Procedure How to prepare a smear from a broth: 1. Aseptically add bacteria to the slide using the inoculating loop. (If the BSL is a 2, then use a sterile wooden stick or disposable loop.) 3 emulsions can fit on the slide, make sure to mix the mix the bacteria and spread the drops out. AVOID any splattering. Flame loop when done 2. Allow the smear to air dry and if prepared correctly the smear should look cloudy. 3. Using a single holder, pass the smear through the upper part of a flame 2-3 times. The heat will fix the preparation. AVOID overheating to decrease the chance of aerosols 4. Allow the slide to cool, and continue with staining. How to prepare a smear from an emulsion: 1. Wearing gloves and eye protection, place a small drop of water on a clean slide using an inoculating loop. 2. Aseptically add bacteria to the slide using the inoculating loop. (If the BSL is a 2, then use a sterile wooden stick or disposable loop.) 3 emulsions can fit on the slide, make sure to mix the mix the bacteria and spread the drops out. AVOID any splattering. Flame loop when done 3. Allow the smear to air dry and if prepared correctly the smear should look cloudy. 4. Using a single holder, pass the smear through the upper part of a flame 2-3 times. The heat will fix the preparation. AVOID overheating to decrease the chance of aerosols 5. Allow the slide to cool, and continue with staining. How to stain a smear: 1. Beginning with a heat fixed emulsion (more than one organism can fit on a slide). 2. Place the slide on a rack over the staining tray. Cover all smears with the stain and make sure any excess in falling into the tray. Make sure to use the methyl blue stain and leave the stain on for 60 seconds before 3. Grasp the slide with a slide holder and hold it at an angle, while GENTLY rinsing the slide with distilled water. Once done dispose the stain tray according to lab practices. 4. Gently blot dry in a tablet of bibulous paper or paper towels. DO NOT RUB, and when dry observe under the oil immersion slide.

ID #7330577

2

SARAH GILDENBERG

LONE STAR COLLEGE - MONTGOMERY

BIOL 2420 - SPRING 2020

Results

Observations and Interpretations Organism or Source

Stain and Duration

Organism #1: Staphylococcus aureus

Methylene Blue for one minute

Organism #2: Escherichia Methylene Blue for one coli minute

Cellular Morphology and Arrangement

Dimensions

Cocci shaped morphology, arranged in staphylo

1000x

Bacilli shaped morphology, with no particular arrangement

1000x

Conclusion Preforming a stain allows us to see the bacteria cells size, shape, and arrangement, although we can see the cells without the stain it is hard to determine these characteristics. When I was looking for my bacteria cells with the Staphylococcus aureus it was difficult to find because they were moving. I did not heat fix the slide long enough. I can improve my technique next time by heat fixing longer.

ID #7330577

3

SARAH GILDENBERG

LONE STAR COLLEGE - MONTGOMERY

BIOL 2420 - SPRING 2020

Follow-Up Questions 1. What are some consequences of leaving a stain on a bacterial smear too long (overstaining)? -Consequences of over-staining are that the cell wall may be broken up or completely destroyed which would result in a loss of morphological characteristics of the bacterial cell. 2. What are some consequences of not leaving a stain on a smear long enough (understaining)? -Some consequences of under-staining are that the cells may lose their stain when washed with water or alcohol. This would make it difficult to identify the cell under the microscope. Consider a coccus and a rod of equal volume. a. Which is more likely to survive in a dry environment? Explain your answer. -Cocci, with their low surface to volume ratio are less efficient at exchange with the environment than rods, but are at an advantage in a dry environment where they lose water dehydrate more slowly than rods. b. Which would be better adapted to a moist environment? Explain your answer. -Organisms with high surface to volume ratio (rods, spirilla) often survive better in moist environments where their ability to exchange materials with their surroundings is an asset for nutrient acquisition of water loss is not a concern.

ID #7330577

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