GENERAL: Microbes, because of their size, are observed by a variety of means. The most obvious method is to magnify the actual organisms to a visual state by which they can be observed. Various types of microscopes have made this possible. The use of many types of stains provides even more information about microbes when they are studied with a microscope. The organisms stain differently based on their particular chemical makeup, and how the retain or do not retain the different stains.

A second method of studying microorganisms, particularly bacteria, fungi, and some parasites, is to grow the microorganisms on artificial media, a substance that provides all the nutrients and enzymes needed for growth. Much like a plant growing from a seed, or a baby from an embryo, the microorganisms multiply substantially, providing a large number of cells in one location which becomes visible to the human eye. This large group of cells is called a colony, and it can be used for additional study of the particular microorganism. The technique of growing these colonies is called "culturing" microorganisms; a single plate is known as a "culture". The colonies can be subjected to different biochemical and enzymatic tests to enable identification of the microorganism.

Today there are many methods available to identify microorganisms at the molecular level, without the need of microscopes or many types of artificial media. These techniques include fatty acid analysis; polymerase chain reactions (PCR) and DNA sequencing. However, these methods are quite expensive and are not used in basic laboratories.

OBJECTIVES: In this laboratory session, you will see examples of how simple stains, as methylene blue, and complex stains, as the Gram stain, are used in microscopic study of microorganisms. You will examine the environmental sampling results for microbes from last week, and come to a conclusion regarding the types and numbers of microbes present and the roles of their presence at a particular site.

INTRODUCTION: Your instructor will demonstrate two staining techniques, and show you examples of the stain and interpretation under a microscope. Depending on time, you may want to try the stains on some of the colonies growing on your environmental culture plates.

EQUIPMENT:

Demonstration:

· Lab Data Sheet, to draw representations of slides that the instructor shows you.
· Pencils

Exercise:

· Your agar plate from last week, with growth from 4 sampled environmental sites.
· 30x microscope (one per every two students) for examination of colonies
· Lab Data Sheet, to record results of observations
· Pencils
· Optional:

Applicator sticks to "pick" from colonies for staining

Clean glass slides
Dropper bottles of distilled water
Gram stain reagents (at sinks)
Methylene blue stain (at sinks)
Large forceps (at sinks)
Paper towels
Microscopes (at demonstration table)
Disposal container at table for contaminated applicator sticks
Squeeze bottles of disinfectants on each table.

1. PROCEDURE:

Demonstration:

1. In the space provided, draw an example of what you see on one of the simple stains. Emphasize any differences in strength of uptake of the stain in any part of the microorganism.

2. In the space provided, draw an example of what you see on one of the examples of the Gram stain. Indicate DIFFERENT COLORS, if seen, where applicable. Be sure to show any differences in shapes (morphology) of the stained organisms.

Exercise:

1. Using your petri plate as a template, draw an outline of your plate on the Lab Data Sheet. Break the drawn plate area into quadrants, as you did with the actual plate last week.

2. Through the TOP of the plate, observe the microbial colonies with your naked eye and with the 30x microscope. On the Data Sheet, draw what you see in one of the quadrants. Using lines and arrow to the different colonies, indicate their color(s) and any other peculiar characteristics you see.

3. Answer the questions on the data sheet pertaining to your cultures.

IF TIME ALLOWS, you may try to stain some of your colonies and look at them through the microscope.

Preparation of slides for staining:

a. Place a small drop of distilled water in the center of one of the slides.
b. Place your agar plate with growth flat on the worksite in front of you.
c. Take one of the applicator sticks. CAREFULLY raise the lid of the petri plate just enough so that you can easily get the stick into the plate but so that it touches only one colony.
d. Touch one... and only one!.... colony with the tip of the stick, and remove the stick without letting it touch anything.
e. Emulsify in the drop of water on the slide some of the organisms that you "picked up" with the stick. Spread the emulsion into a thin layer over about 3/4 by 3/4 inch on the slide. Allow to air dry.
f. When the slide is air dry, take it to your instructor, who will "heat fix". The slide with microorganisms is now ready to stain.
g. Decide which stain you are going to use, and follow the instructions printed on the placards at the sinks.
h. When you are completed with the stain, carefully blot excess water off of the slide using a paper towel. Allow the slide to air dry.
i. Take the slide to your instructor, who will focus it under one of the microscopes.
j. On the Lab Data sheet, draw a picture of the bacteria or fungus that you see. Note colors; variations.

Dispose of all plates and applicator sticks, into the biohazard waste bags found at the end of your table.

QUESTIONS: Environmental Cultures

1. Did you have growth in every quadrant of your plate? If not, explain why you do not think anything grew.

2. What area that you tested surprised you by whether it had microbes or not? Can you think of an explanation for the results?

3. Which type of microorganism was predominant in the area on your plate that you studied: bacteria (generally smooth or crinkled small colonies, white to gray to tan) or fungi (frequently large "spreading" colonies, fluffy or with a mold-like appearance)?

4. Give two reasons why it is important to keep the lids on Petri plates with microorganisms growing on them. Why should the lids be kept on them before they are inoculated?

5. Why don’t we allow eating, drinking, smoking or applying of cosmetics in a microbiology laboratory?

1. DEMONSTRATION EXAMPLES

Simple Stain Gram Stain

2. ENVIRONMENTAL CULTURE RESULTS
(Use Petri Plate as a pattern to draw the plate)

Optional Staining Exercise: Microorganisms from Environmental Cultures

Draw examples of what you see on the slides prepared from your environmental culture. Indicate colors and morphology that are observed.

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