DEPARTMENT OF MICROBIOLOGY
FACULTY OF NATURAL AND APPLIED SCIENCES
UMARU MUSA YARADUA UNIVERSITY KATSINA
BASIC TECHNIQUES IN MICROBIOLOGY (MCB 231)
CULTURING OF MICROORGANISMS
Refers to the process of growing microorganisms under controlled conditions, usually in a
laboratory setting, to study their properties, behavior, and characteristics.
Culture Media
Culture media (Growth media) are substances or mixtures used to grow and support the growth of
microorganisms, cells, or tissues in laboratory settings. They provide the necessary conditions
(such as pH, temperature, and oxygen) for the organisms to grow and reproduce.
They are classified on the basis of several parameters:
i. Physical Nature
ii. Chemical constituent from which they are made
iii. Function
Types of Media
1. Physical Nature
a. Liquid media (Broth)
No solidifying agent (like agar)
Example: Nutrient broth
Use: Growing large number of microorganisms, observing metabolic activity
Appearance: Clear or slightly cloudy when growth occurs
b. Solid Media
Contains higher agar concentration (1.5- 2%)
Example: Nutrient agar
Use: Isolation of pure colonies, studying colony morphology
Texture: Firm surface for microbial growth
c. Semi-solid Media
Contains a small amount of agar (0.2-0.5%)
Example: Motility test medium
Use: Testing bacterial motility and oxygen requirements
Texture: Soft, jelly like
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2. Chemical Composition
a. Synthetic (chemically defined) Media
Exact chemical composition is known
Example: Glucose salt medium
Use: Research purposes where precise nutrient control is needed
b. Undefined (Complex) Media
Composition not exactly known (contains extract like yeast or beef)
Example: Nutrient agar, blood agar
Use: General microbial growth
3. Functional Type
a. Nutrient Media (General purpose)
Support growth of many organisms
Example: Nutrient agar
Use: Routine cultivation
b. Enrichment Media
Contains extra nutrients (blood, serum, egg)
Example: Blood agar
Use: Growing fastidious organisms ( organisms with complex nutritional needs)
c. Selective Media
Contains substances that inhibit some organisms but allow others
Example: MacConkey agar(selects Gram- negative bacteria)
Use: Isolation of specific groups
d. Differential Media
Distinguishes organisms based on biochemical reactions
Example: MacConkey agar (lactose fermentation, color change)
Use: Identification of bacteria
e. Transport Media
Maintains viability of organisms during transport
Example: Staurts transport medium
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Use: Clinical transport medium
Preparation of Culture Media
Culture medium (agar, broth, or slants) is prepared and sterilized, typically through
autoclaving, to eliminate any unwanted microorganism
Media is dispensed in a sterile container using aseptic technique.
Inoculation
Is the process of introducing microorganisms into a sterile culture medium (agar plates,
slants, or liquid broth) by streaking, stabbing, pouring or pipetting, depending on the
desired outcome
Bacteria grow on solid or liquid media while Fungi is similar to bacteria, but may require
more specialized media (e.g Sabouraud dextrose agar, potato dextrose agar).
Viruses require living cells for replication and are cultured in cell cultures, embryonated
eggs, or animals
Incubation of Culture
Once microorganisms are cultured or isolated, proper incubation conditions (temperature,
humidity, oxygen, pH and time) must be maintained to encourage optimal growth.
Temperature: Incubator provide a controlled environment with constant temperature
depending on the temperature requirement of microorganisms (e.g., 37°C for human
pathogens, 25°C to 30°C for Fungi).
Time: the length of incubation depend on the type of microorganisms and the growth rate.
Some organisms may show visible growth in a few hours, while others may take days to
form colonies or reach detectible levels in liquid media.
Oxygen Availability: Aerobic (require oxygen), Anaerobic (grow in the absence of
oxygen), Facultative anaerobes (can grow with or without oxygen), and Microaerophilic
organisms (require low oxygen levels).
Humidity: some microorganisms require a humid environment to prevent the media from
drying out, particularly in solid media plate
Isolation of Microorganism
Isolation is the technique of separating a single species of microorganism from a mixed culture,
ensuring that only one type of microorganism is present in the culture.
a. Bacteria: Methods of isolation include streak plate method, pour plate or spread plate
b. Fungi: can be isolated by direct plating, streak plate method, pour plate method or spread plate
c. Viruses: isolation can be achieved through filtration, centrifugation
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Common Isolation Methods
1. Streak Plate Method
Procedure
A sterile loop is dipped into the sample
The loop is streaked across one section of agar plate
The loop is sterilized and streaked again across another section
The loop is then reflamed, and streaked once again in the third area.
Repeated streaking reduces microbial density
Result
Discrete colonies develop from single cells.
Advantages
Simple and widely used
Does not require special equipment
Disadvantages
Requires practice for proper isolation
2. Spread Plate Method
Procedure
Prepare serial dilutions of sample
Transfer a small volume onto agar plate
Spread using sterile spreader (glass rod)
Result
Colonies form on the surface.
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Advantages
Useful for counting viable cells
Even distribution
Disadvantages
Only small sample volume can be used
3. Pour Plate Method
Procedure
Pipette a measured amount of the microbial sample into the sterile petri dish
Pour the cooled molten agar (~45°C) into the plate containing the sample
Swirl the plate carefully to mix the sample evenly with the agar
Leave the plate undisturbed until the agar solidifies
Result
Colonies develop both within the agar and on the surface.
Advantages
Good for counting bacteria
Suitable for anaerobic/microaerophilic organisms
Disadvantages
Heat may kill some microbes
Colonies inside agar are harder to pick
Pure Culture Techniques
After isolation a pure culture is established, a pure culture contains only a single species of
microorganism. Obtaining a pure culture is critical for accurate identification and study of
microbial characteristics.
Sub-culturing: After isolating a colony on an agar plate, a small portion of the colony is
transferred to a fresh medium to ensure that the culture remains pure.
Re-streaking: If mixed growth occurs, a colony is restreaked onto a fresh plate to ensure
isolation.
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Identification of Isolated Microorganisms
Once microorganisms are isolated, they are identified based on their morphological, biochemical,
and genetic characteristics.
a. Bacteria: Bacterial species are identified based on colony morphology (size, shape, color etc.),
gram stain reaction, biochemical tests (catalase, oxidase, indole etc.) and molecular
techniques
b. Fungi: Fungal identification is based on colony morphology, microscopic examination
(hyphae, conidia, spores), biochemical tests (carbohydrate utilization test, urease etc.)and
molecular techniques
c. Viruses: Identification is often done via, electron microscopy, immunofluorescence, Elisa or
molecular techniques
Preservation of Cultures
Refers to the techniques used to maintain the viability and stability of microbial cultures for
research, industrial applications, or medical purposes.
Short Term Storage: Refrigeration (40c), Agar slants and stabs, Glycerol stock, Dessication.
Long Term Storage: Cryopreservation (freezing), Lyophilization (freeze-drying).
STAINING TECHNIQUES
Staining involves the application of dyes (also known as stains) to biological specimens, typically
microorganisms, to enhance their visibility under a microscope. Stains bind to specific components
of the cells, such as the cell wall, cytoplasm, or nucleus, providing contrast against the background.
Importance of Staining
Enhances Visibility: Increases the contrast between microorganisms and the background.
Differentiates Microorganisms: Some stains differentiate microorganisms based on their
structural differences (e.g., Gram staining).
Identifies Cellular Components: Can help identify cellular structures such as cell walls,
nuclei, and flagella.
Classifies Organisms: Staining techniques such as Gram staining help classify bacteria
into groups (Gram-positive and Gram-negative).
Helps in Pathogen Detection: Crucial in clinical microbiology for identifying pathogens
in patient samples.
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Basic Principles of Staining
1. Dyes: Chemical compounds that impart color to the specimen.
Basic Dyes: Positively charged dyes (e.g., crystal violet, methylene blue) that bind to
negatively charged components like nucleic acids and cell walls.
Acidic Dyes: Negatively charged dyes (e.g., eosin, nigrosin) that bind to positively charged
components, often used in negative staining techniques.
2. Fixation: The process of preparing and preserving the sample. It usually involves using heat or
chemicals (e.g., formaldehyde) to kill and "fix" the specimen to the slide.
3. Staining process:
Staining can be achieve through the following processes:
Using a single dye to enhance the contrast and shape of the microorganism.
Using two or more stains to differentiate organisms based on their chemical or structural
differences.
Types of Staining Techniques
1. Simple staining
2. Differential staining
3. Special staining
1. Simple Staining
Purpose: Uses a single dye to enhance the contrast, shape, size, and arrangement of
microorganisms. Simple stain can be classified into two types:
a. Positive (Direct) staining: the stain directly binds to the cell or structure of interest
Procedure:
A small amount of microorganism is smeared onto a microscope slide and allowed to air
dry.
The slide is then heat-fixed by passing it through a flame to kill and adhere the
microorganism.
A single dye (e.g., methylene blue, crystal violet) is applied to the slide and left for a few
minutes.
The slide is rinsed with water, dried, and observed under a microscope.
Common Stains: Methylene blue, crystal violet, safranin.
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Outcome: Provides color to the microorganisms, making them easier to visualize, but does not
differentiate between types of microorganisms.
b. Negative (Indirect ) staining: the background is stained, leaving the cell unstained
Procedure:
Place a small drop of acidic dye (e.g., nigrosin, indian ink) on the edge of a clean
microscope slide.
Add a small amount of the bacterial culture to the drop.
Use another slide to spread the drop across the slide at 30-450 angle.
Allow the slide to air dry and view the slide under microscope, starting with low
magnification and increasing to high magnification.
Common Stains: Nigrosin, indian ink, eosin.
Outcome: The background is darkly stained, and the bacterial cells appear as clear or white
structures against the dark background.
2. Differential Staining
These techniques involve the use of more than one stain to differentiate organisms or their cellular
components based on specific characteristics. Some examples include:
a. Gram Staining
Purpose: To classify bacteria into Gram-positive and Gram-negative based on the characteristics
of their cell walls.
Procedure:
Prepare a smear of bacterial culture on a slide and fix it.
Flood the slide with crystal violet (primary stain) for about 1 minute.
Rinse the slide with water and apply iodine solution (mordant) for 1 minute to form a
crystal violet-iodine complex.
Rinse with alcohol or acetone (decolorization step). Gram-positive bacteria retain the
purple color, while Gram-negative bacteria lose it.
Apply safranin (counterstain) for 30 seconds. Gram-negative bacteria take up the safranin
and appear pink, while Gram-positive bacteria retain the purple crystal violet color.
Rinse, dry, and observe under a microscope.
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Outcome:
Gram-positive bacteria: Retain the crystal violet stain and appear purple due to their thick
peptidoglycan layer.
Gram-negative bacteria: Take up the safranin and appear pink due to their thinner
peptidoglycan layer and outer membrane.
b. Acid-Fast Staining
Purpose: To identify acid-fast bacteria (e.g., Mycobacterium tuberculosis) which have a waxy
lipid layer (mycolic acid) in their cell walls that prevents staining with conventional methods.
Procedure:
Prepare and heat-fix a smear of the specimen on a slide.
Apply carbol fuchsin (primary stain) and heat for 5 minutes to penetrate the waxy cell
wall.
Rinse with water, then decolorize with acid-alcohol.
Counterstain with methylene blue.
Rinse with water, dry, and observe under a microscope.
Outcome:
Acid-fast bacteria: Retain the red color of carbol fuchsin.
Non-acid-fast bacteria: Appear blue due to the methylene blue counterstain.
3. Special Staining
Refers to a group of advanced laboratory techniques used to highlight specific structures,
components or type of microorganism within biological sample. Some of these methods include:
a. Endospore Staining
Purpose: To visualize bacterial endospores (e.g., Bacillus and Clostridium species), which are
resistant structures formed under unfavorable conditions.
Procedure:
Prepare a smear of the bacterial culture and heat-fix it.
Apply malachite green (primary stain) and heat the slide to force the stain into the spores.
Rinse with water to remove excess stain.
Counterstain with safranin for 30 seconds.
Rinse, dry, and observe under a microscope.
Outcome:
Endospores: Stain green due to malachite green.
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Vegetative cells: Stain red due to safranin.
b. Capsule Staining
Purpose: To visualize bacterial capsules, which are protective structures made of polysaccharides
or proteins.
Procedure:
Prepare a smear of the sample without heat-fixing to preserve the capsule.
Stain the background with nigrosin (an acidic dye).
Stain the bacterial cells with crystal violet (a basic dye).
Rinse gently with water, being careful not to disrupt the capsule.
Dry and observe under a microscope.
Outcome:
Capsules: Appear as clear halos around the bacterial cells.
Bacterial cells: Appear purple, while the background is dark.
c. Flagella Staining
Purpose: To observe bacterial flagella, which are responsible for motility.
Procedure:
Prepare a thin smear of bacteria and air-dry it.
Apply a special flagella stain (e.g., Leifson’s flagella stain) that binds to flagella and
increases their thickness.
Observe under the microscope.
Outcome: Flagella are stained and appear as fine structures emerging from the bacterial cells.
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