Lesson 3:

The Methods for Studying Microorganisms


Objective

In this lesson we will learn the following concepts:

  • Microbes are managed and characterized by using the Five I's (inoculation, incubation, isolation, inspection and identification).
  • Various cultures and media are used in studying microorganisms.
  • The parts of a microscope and how they are used for magnifying cells and their parts.
  • The different staining procedures for viewing cells.

 


Reading Assignment

Along with the online lecture, read Chapter 2 in Wastewater Microbiology.





Lecture

Introduction

Growing up you were probably told to wash your hands so you would not become infected by germs. You probably only did so to stay out of trouble because no matter how well you focused on your hands, you never saw a germ on them. Today you realize that a germ is a microorganism, one of many that surround us. Microorganisms cannot be seen with the naked eye, but you can see them with the aide of a microscope. In this lesson you'll learn how to view microorganisms under a microscope.

 

 

The Five I's

The Five I's is a method used to locate, grow, observe and characterize microorganisms. It is not necessary to perform all of these steps, or to perform them exactly in this order. In some cases, one may proceed right from the sample to inspection, and in others, only inoculation and incubation on special media are required. The first step is the collect your specimen. Common specimens are body fluids, foods, water or soil.

One you have your specimen collected you can perform the Five I's:

  1. Inoculation: The sample is placed into a container of sterile medium that provides microbes with the appropriate nutrients to sustain growth.



  1. Incubation: An incubar can be used to adjust the proper growth conditions of a sample.



  2. Isolation: The end result of inoculation and incubation is isolation of the microbe.



  3. Inspection: The cultures are observed for obvious growth characteristics that could be useful in analyzing the specimen contents.



  4. Identification: Determine the type of microbe, usually to the level of species.

     

 

The Microscope

The principal way a microbiologist studies microorganisms is by observing them through a microscope. A microscope is a device that enlarges objects using a process called magnification. The simplest form of a microscope is a magnifying glass consisting of a single lens. And the simplest magnifying glass is the bottom of an empty glass. Some glasses are slightly bent at the bottom, causing a magnifying effect if held at a certain height over an object.

 

The single-lens magnifying lens or glass is a thing of the past. Scientists today use a microscope that has two sets of lenses (objective and ocular), which is called a compound light microscope. View different slides of organisms from algae to bacteria.

 

A light compound micrscope is a good tool for observing many kinds of microorganisms, however, it isn't capable of seeing the internal structure of a microorganism nor can it be used to observe a virus. In order to view internal structures of viruses and microorganisms, microbiologists use an electron microscope where specimens are viewed in a vacuum. The electron microscope uses beams of electrons and magnetic lenses rather than light waves and optical lenses to view a specimen.

 

 


Providing Nutrients in the Laboratory

In order to grow microbes out of their natural habitats and in pure form in the laboratory, certain nutrients are required. Nutritional requirements of microbes vary, and at least 500 different types of media are used in culturing and identifying microorganisms. Culture media are contained in test tubes, flasks, or Petri dishes. Media are extremely varied in nutrient content and consistency and can be specially formulated for a particular purpose. There are different levels of media such as liquid, semisolid, and solid. The most widely used media is agar, a complex polysaccharide. The benefits of using agar as the media include that is is solid at room temperature, and melts at the boiling temperature of water. It is flexible and moldable and provides a basic framework to hold moisture and nutrients, though it is not itself a digestible nutrient for most microorganisms.

 

 

Preparing Specimens

There are two ways to prepare a specimen to be observed under a light compound microscope. These are a smear and a wet mount.

 

Smear

A smear is a preparation process where a specimen is spread on a slide. You prepare a smear using the heat fixation process:

  1. Use a clean glass slide.
  2. Take a loop of the culture.
  3. Place the live microorganism on the glass slide.
  4. The slice is air dried then passed over a Bunsen burner about three times.
  5. The heat causes the microorganism to adhere to the glass slide. This is known as fixing the microorganism to the glass slide.
  6. Stain the microorganism with an appropriate stain.

 

 

Wet Mount

A wet mount is a preparation process where a live specimen in culture fluid is placed on a concave glass slide or a plain glass slide. The concave portion of the glass slide forms a cup-like shape that is filled with a thick syrupy substance. The microorganism is free to move about within the fluid, although the viscosity of the substance slows its movement. This makes it easier for you to observe the microorganism. The specimen and the substance are protected from spillage and outside contaminants by a glass cover that is placed over the concave portion of the slide.

 

 

Staining a Specimen

Not all specimens can be clearly seen under a microscope. Sometimes the specimen blends with other objects in the background because they absorb and reflect the same light waves. You can enhance the appearance of a specimen by using a stain. A stain is used to contrast the specimen from the background.

A stain is a chemical that adheres to structures of the microorganism and in effect dyes the microorganism so that it can be easily seen under a microscope. Stains used in microbiolody are either basic or acidic.

Basic stains are cationic and have a positive charge. Common basic stains are methylene blue and crystal violet. These are ideal for staining chromosomes and the cell membranes of many bacteria.

Acid stains are anionic and have a negative charge. Common acidic stains are eosin and picric acid. Acid stains are used to stain cytoplasmic material and organelles or inclusions.

There are two types of stains: simple and differential. A simple stain has a single basic dye that is used to show shapes of cells and structures within a cell.

A differential stain consists of two or more dyes and is used in the procedure to identify bacteria. One of the most commonly used differential stains is the gram stain. Gram-positive microorganisms stain purple. Gram-negative microorganisms stain pink. A common bacterium that causes food poisoning, Staphylococcus aureus, is gram-positive. Escherichia coli is gram-negative. Read about the gram-stain procedure.

 

Review

The Five I's is a method used to locate, grow, observe and characterize microorganisms which include inoculation, incubation, isolation, inspection and identification. The principal way a microbiologist studies microorganisms is by observing them through a microscope, either a compound light or electron. In order to grow microbes out of their natural habitats and in pure form in the laboratory, certain nutrients are required. The most widely used media is agar. There are two ways to prepare a specimen to be observed under a light compound microscope: a smear or a wet mount. Not all specimens can be clearly seen under a microscope. You can enhance the appearance of a specimen by using a stain.

 

 

Source

Betsy, Dr. Tom and Keogh, Jim. 2005. Microbiology Demystified . McGraw-Hill Publishing

 

Assignments

Complete the interactive exercises in Assignment 3

This assignment will give you practice with the topics covered in Lesson 3.  You should print the assignment and become familiar with the exercises before doing them online. You may do the Assignment online to get credit or print it out and send it to the instructor.  It will require the Flash player to view, which should already be installed on your machine.

 

 

Lab

Please read the Laboratory Microscope and the Gram Stain lab procedures. No work needs to be done in regards to this lab.

 

 

Quiz

Answer the questions in the Lesson 3 Quiz .  When you have gotten all the answers correct, print the page and either mail or fax it to the instructor. You may also take the quiz online and directly submit it into the database for a grade.