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  Section: Biotechnology Methods » Microbiology
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  The Microscopy
  The Bright Field Microscope
  Introduction to the Microscope and Comparison of Sizes and Shapes of Microorganisms
  Cell Size Measurements: Ocular and Stage Micrometers
  Measuring Depth
  Measuring Area
  Cell Count by Hemocytometer or Measuring Volume
  Measurement of Cell Organelles
  Use of Darkfield Illumination
  The Phase Contrast Microscope
  The Inverted Phase Microscope
  Aseptic Technique and Transfer of Microorganisms
  Control of Microorganisms by using Physical Agents
  Control of Microorganisms by using Disinfectants and Antiseptics
  Control of Microorganisms by using Antimicrobial Chemotherapy
  Isolation of Pure Cultures from a Mixed Population
  Bacterial Staining
  Direct Stain and Indirect Stain
  Gram Stain and Capsule Stain
  Endospore Staining and Bacterial Motility
  Enumeration of Microorganisms
  Biochemical Test for Identification of Bacteria
  Triple Sugar Iron Test
  Starch Hydrolysis Test (II Method)
  Gelatin Hydrolysis Test
  Catalase Test
  Oxidase Test
  IMVIC Test
  Extraction of Bacterial DNA
  Medically Significant Gram–Positive Cocci (GPC)
  Protozoans, Fungi, and Animal Parasites
  The Fungi, Part 1–The Yeasts
  Performance Objectives
  The Fungi, Part 2—The Molds
  Viruses: The Bacteriophages
  Serology, Part 1–Direct Serologic Testing
  Serology, Part 2–Indirect Serologic Testing

Laboratory Rules
For the safety and convenience of everyone working in the laboratory, it is important that the following laboratory rules be observed at all times:
  1. Place only those materials needed for the day’s laboratory exercise on the benchtops.
  2. Since some of the microorganisms used in this class are pathogenic or potentially pathogenic (opportunistic), it is essential to always follow proper aseptic technique in handling and transferring all organisms.
  3. No eating, drinking, or any other hand-to-mouth activity while in the lab. If you need a short break, wash your hands with disinfectant soap and leave the room.
  4. Using a wax marker, properly label all inoculated culture tubes or petri plates with the name or initials of the microorganism you are growing, your initials or a group symbol, and any other pertinent information. Place all inoculated material only on your assigned incubator shelf. Culture tubes should be stored upright in plastic beakers, while petri plates should be stacked and incubated upside-down.
  5. Always clean the oil off of the oil immersion lens of the microscope with a piece of lens paper at the completion of each microscopy lab.
  6. Disinfect the benchtop with isopropyl alcohol before and after each lab period. Be sure your Bunsen burner is turned off before you spray any alcohol.
  7. Always wash your hands with disinfectant soap.

General Directions
  1. Familiarize yourself in advance with the procedure of the experiment to be performed.
  2. Disinfect the working table with isopropyl alcohol before and after each lab.
  3. The first part of each lab period will be used to complete and record the results of previous experiments. We will always go over these results as a class. You may wish to purchase a set of colored pencils to aid you in recording your results in the lab manual.
  4. Wash your hands with disinfectant soap before leaving the lab.
Binomial Nomenclature
Microorganisms are given specific scientific names based on the binomial (2 names) system of nomenclature. The first name is referred to as the genus and the second name is termed the species. The names usually come from Latin or Greek and describe some characteristic of the organism.

To correctly write the scientific name of a microorganism, the first letter of the genus should be capitalized, while the species name should be in lowercase letters. Both the genus and species names are italicized or underlined. Several examples are given below:

Bacillus subtilus
Bacillus: L. dim. noun Bacillum, a small rod
subtilus: L. adj. subtilus, slender

Escherichia coli
Escherichia: after discoverer, Prof. Escherich
coli: L. gen. noun coli, of the colon

Staphylococcus aureus
Staphylococcus: Gr. noun Staphyle, a bunch of grapes; Gr. noun coccus, berry
aureus: L. adj. aureus, golden

Metric Length and Fluid Volume
The study of microorganisms necessitates an understanding of the metric system of length. The basic unit of length is the meter (m), which is approximately 39.37 inches. The basic unit for fluid volume is the liter (L), which is approximately 1.06 quarts. The prefix placed in front of the basic unit indicates a certain fraction or multiple of that unit. The most common prefixes we will be using are:

centi = 10–2 or 1/100
centimeter (cm) = 10–2 m or 1/100 m
milli = 10–3 or 1/1000
millimeter (mm) = 10–3 m or 1/1000 m
milliliter (mL) = 10–3 L or 1/1000 L
micro = 10–6 or 1/1,000,000
micrometer (µm) = 10–6 m or 1/1,000,000 m
microliter (µL) = 10–6 L or 1/1,000,000 L
nano = 10–9 or 1/1,000,000,000
nanometer (nm) = 10–9 m or 1/1,000,000,000 m.

In microbiology, we deal with extremely small units of metric length (micrometer, nanometer). The main unit of length is the micrometer (mm), which is 10–6 (1/1,000,000) of a meter or approximately 1/25,400 of an inch.

The average size of a rod-shaped (cylindrical) bacterium is 0.5–1.0 mm wide by 1.0–4.0µm long. An average coccus-shaped (spherical) bacterium is about 0.5–1.0 µm in diameter. A volume of 1 cubic inch is sufficient to contain approximately 9 trillion average-sized bacteria. It would take over 18,000,000 average-sized cocci lined up edge-to-edge to span the diameter of a dime. In several labs, we will be using pipettes to measure fluid volume in mL.


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