Ch. Methods for Studying Microorganisms
Culturing microorganisms Microscopy Staining
Culturing microorganisms
Microscopy
Staining
You will be exposed to much of the information in this chapter in the laboratory part of this course. We will only cover a few topics in lecture.
Culturing Microorganisms in the Laboratory
Culturing (growing) a microorganism involves:
1. Inoculation of a suitable growth medium (solid or liquid) with the microorganism or a specimen containing the microorganism
The medium provides a source of nutrients needed by the cell to grow Ex. nutrient broth, tryptic soy agar
The medium provides a source of nutrients needed by the cell to grow
Ex. nutrient broth, tryptic soy agar
2. Incubation for a period of time using conditions that allow the organism to grow
Ex. 48 hours, 37OC, no O2 (anaerobic)
Different microorganisms have different nutritional requirements
Some microorganisms have simple requirements
Ex. CO2 (source of carbon), H2 (source of energy) Nitrogen, Phosphorus and minerals
Some are fastidious and have complex requirements
Ex. vitamins, amino acids, lipids
Types of media (based on chemical composition)
1. Synthetic (chemically defined)
All chemicals and their exact amount are known Pure chemicals are used and precisely weighed
All chemicals and their exact amount are known
Pure chemicals are used and precisely weighed
2. Complex
The exact chemical composition is not known Complex substances provide the nutrients Ex. blood, yeast extract, vegetable or meat broth
The exact chemical composition is not known
Complex substances provide the nutrients
Ex. blood, yeast extract, vegetable or meat broth
Simple microscope
Invented by Leeuwenhoek (Fig. 1.9) Single lens ~300 X magnification 1676 He reported the discovery of bacteria and other microorganisms after observing them in rainwater in which crushed pepper had been soaked He also observed them in many other samples such as the scrappings from his teeth
Invented by Leeuwenhoek (Fig. 1.9)
Single lens ~300 X magnification 1676 He reported the discovery of bacteria and other microorganisms after observing them in rainwater in which crushed pepper had been soaked
He also observed them in many other samples such as the scrappings from his teeth
Compound microscope (Fig. 3.14)
Uses two lenses to magnify the image of a specimen (Fig. 3.15) 1. Objective. Contains the first lens that magnifies the image of the specimen Low power objective. 10X magnification High dry power objective. 40X magnification Oil immersion objective. 100X magnification (Fig. 3.17) Oil is used to increase the amount of light that enters the objective by decreasing light refraction 2. Ocular. Contains a lens that magnifies the image further, usually 10X Total magnification = Objective x Ocular Ex. 100X x 10X = 1000X when using the oil immersion objective Condenser. Concentrates light on specimen Diaphragm. Regulates amount of light Stage. Supports the glass slide that contains the specimen
Uses two lenses to magnify the image of a specimen (Fig. 3.15)
1. Objective. Contains the first lens that magnifies the image of the specimen
Low power objective. 10X magnification High dry power objective. 40X magnification Oil immersion objective. 100X magnification (Fig. 3.17) Oil is used to increase the amount of light that enters the objective by decreasing light refraction
Low power objective. 10X magnification
High dry power objective. 40X magnification
Oil immersion objective. 100X magnification (Fig. 3.17)
Oil is used to increase the amount of light that enters the objective by decreasing light refraction
2. Ocular. Contains a lens that magnifies the image further, usually 10X
Total magnification = Objective x Ocular Ex. 100X x 10X = 1000X when using the oil immersion objective
Total magnification = Objective x Ocular
Ex. 100X x 10X = 1000X when using the oil immersion objective
Condenser. Concentrates light on specimen
Diaphragm. Regulates amount of light
Stage. Supports the glass slide that contains the specimen
Staining microorganisms
Two main purposes. 1. To make microorganisms and the components of their cells easier to see with a light microscope Unstained cells have poor contrast and are difficult to see Unstained cell structure are hard to see Ex. flagella, capsules, endospores 2. Classification and taxonomic identification of microorganisms
Two main purposes.
1. To make microorganisms and the components of their cells easier to see with a light microscope
Unstained cells have poor contrast and are difficult to see Unstained cell structure are hard to see Ex. flagella, capsules, endospores
Unstained cells have poor contrast and are difficult to see
Unstained cell structure are hard to see
Ex. flagella, capsules, endospores
2. Classification and taxonomic identification of microorganisms
Types of stains (Fig. 3.26)
1. Simple stains. Single dye is used to stain cells Ex. methylene blue Shows shape and arrangement of cells 2. Differential stains. Two or more dyes are used Used to distinguish between different types of microorganisms -Gram stain A differential stain that distinguishes between two major groups of bacteria Gram positive organisms stain purple Gram negative organisms stain red (Old cells may be variable and not give an accurate result)
1. Simple stains. Single dye is used to stain cells
Ex. methylene blue Shows shape and arrangement of cells
Ex. methylene blue
Shows shape and arrangement of cells
2. Differential stains. Two or more dyes are used
Used to distinguish between different types of microorganisms -Gram stain A differential stain that distinguishes between two major groups of bacteria Gram positive organisms stain purple Gram negative organisms stain red (Old cells may be variable and not give an accurate result)
Used to distinguish between different types of microorganisms
-Gram stain
A differential stain that distinguishes between two major groups of bacteria Gram positive organisms stain purple Gram negative organisms stain red (Old cells may be variable and not give an accurate result)
A differential stain that distinguishes between two major groups of bacteria
Gram positive organisms stain purple Gram negative organisms stain red
(Old cells may be variable and not give an accurate result)
-Acid-fast stain A differential stain for Mycobacteria (some cause tuberculosis --Mycobacterium tuberculosis and leprosy --Mycobacterium leprae) Mycobacteria are acid-fast and stain red Non-acid-fast bacteria stain blue
-Acid-fast stain
A differential stain for Mycobacteria (some cause tuberculosis --Mycobacterium tuberculosis and leprosy --Mycobacterium leprae) Mycobacteria are acid-fast and stain red Non-acid-fast bacteria stain blue
A differential stain for Mycobacteria (some cause tuberculosis --Mycobacterium tuberculosis and leprosy --Mycobacterium leprae)
Mycobacteria are acid-fast and stain red
Non-acid-fast bacteria stain blue
3. Special stains. Used to see specialized cell structures Endospore stain for spores inside cells Flagella stain Negative stain to visualize capsules surrounding cells
3. Special stains. Used to see specialized cell structures
Endospore stain for spores inside cells Flagella stain Negative stain to visualize capsules surrounding cells
Endospore stain for spores inside cells
Flagella stain
Negative stain to visualize capsules surrounding cells
End of Chapter 3 notes.
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SIUC / College of Science / Microbiology / Microbiology 201 http://www.micro.siu.edu/micr201/chapter3N.html Last updated: Jan 30, 2005 /jdh