Microbiological Media and Culture Techniques
General Media Classification
All microbiological media can be categorized into four primary groups, each serving a specific purpose for bacterial growth and identification.
Primary (General/Isolation) Media
Primary media, also known as general or general isolation media, is designed to support the growth of most non-fastidious bacteria. These bacteria are considered 'not picky,' meaning they do not require specialized or rare nutrients to thrive. This type of media contains general nutrients sufficient for broad bacterial growth.
Enrichment Media
Enrichment media is formulated with added nutrients to support the growth of fastidious or 'picky' bacteria. These bacteria require specific, often complex, growth factors. Common additives include blood serum, egg yolk, or specific proteins essential for the bacteria's survival.
Selective Media
Selective media is engineered to encourage the growth of a desired microbe while simultaneously suppressing or inhibiting the growth of unwanted bacteria. For example, if a urine sample contains both E. coli and Staphylococcus, a selective medium can be chosen that provides nutrients only to E. coli while containing agents that inhibit the growth of Staphylococcus.
Differential Media
Differential media allows for the visual differentiation of bacterial species, typically through a color change. This color change occurs depending on the metabolic characteristics of the bacteria growing on the plate. For instance, some bacteria might cause the media to turn neon green, indicating a specific species like E. coli.
Specific Types of Media
Trypticase Soy Agar (TSA) / Nutrient Agar (NA)
Type: General purpose (primary) media.
Purpose: Grows a wide variety of microbes as long as they are non-fastidious (not picky). It is not selective for any particular type of microbe; it supports the growth of many different species.
Composition: Complex media containing many ingredients to support diverse life. It grows both gram-positive and gram-negative bacteria.
Broth Version: Trypticase Soy Broth (TSB) or Nutrient Broth (NB) are liquid versions of the agar.
Visual: Colonies appear as clusters of thousands of cells, originating from a single bacterial cell that multiplied extensively.
Phenylethyl Alcohol Agar (PEA)
Type: Selective media.
Purpose: Selects for the growth of gram-positive bacteria only. It does not allow gram-negative bacteria to grow.
Mechanism: Contains phenylethyl alcohol and other chemicals that kill or inhibit the growth of gram-negative bacteria.
Identification: If growth is observed on PEA agar, the bacteria are confirmed to be gram-positive.
MacConkey Agar (MAC)
Type: Selective and differential media.
Selective Purpose: Selects for the growth of gram-negative bacteria. It contains chemicals that inhibit or kill gram-positive bacteria.
Differential Purpose: Differentiates gram-negative bacteria based on their ability to ferment lactose.
Lactose Fermenters: If gram-negative bacteria can consume and break down the lactose sugar present in the media, they produce an acid byproduct. This acid causes a color change, turning the colonies and surrounding agar a pinkish-red color.
Non-Lactose Fermenters: If gram-negative bacteria cannot eat lactose, they do not produce acid, and no color change occurs. The colonies appear white or yellow/clear.
Lactose: A sugar included in the agar that serves as a food source for bacteria capable of fermenting it.
Identification: Growth on MAC confirms gram-negative bacteria. Pink/red colonies indicate a lactose-fermenting gram-negative, while clear/yellow/white colonies indicate a non-lactose-fermenting gram-negative.
Mannitol Salt Agar (MSA)
Type: Selective and differential media.
Selective Purpose: Selects for the growth of Staphylococcus bacteria only. Other bacteria, like E. coli, will not grow on this medium.
Mechanism: Contains a high salt concentration of approximately 7.5 ext{%}. Most bacteria cannot survive this osmotic pressure, as salt draws water out of their cells. Staphylococcus species, however, are halotolerant and can withstand high salt environments.
Differential Purpose: Differentiates Staphylococcus species based on their ability to ferment mannitol.
Staphylococcus aureus: This species can ferment (eat/break down) the mannitol sugar in the agar. This process produces an acid byproduct, which causes the agar to turn yellow (a bright neon color) around the colonies.
Other Staphylococcus species: These species cannot ferment mannitol. They do not produce acid, and thus no color change occurs. The agar remains its initial pinkish-red color.
Mannitol: A sugar included in the agar to test for fermentation.
Identification: Growth on MSA confirms Staphylococcus. Yellow growth indicates Staphylococcus aureus, while pink/red growth indicates other Staphylococcus species.
Eosin Methylene Blue (EMB) Agar
Type: Selective and differential media.
Selective Purpose: Selects for the growth of gram-negative bacteria. It contains chemicals that inhibit or kill gram-positive bacteria.
Differential Purpose: Differentiates gram-negative bacteria based on strong or weak lactose fermentation.
E. coli: A strong lactose fermenter, E. coli produces a significant amount of acid, resulting in colonies with a characteristic metallic green sheen or a metallic bluish-black color.
Other Lactose Fermenters: Some gram-negative bacteria ferment lactose but less vigorously than E. coli, leading to pink or reddish colonies without a metallic sheen.
Non-Lactose Fermenters: Gram-negative bacteria that do not ferment lactose will appear colorless or translucent, taking on the color of the media.
Identification: Growth confirms gram-negative bacteria. Metallic green colonies specifically identify E. coli. Pink colonies indicate other gram-negative lactose fermenters, and colorless colonies indicate non-lactose fermenting gram-negatives.
Blood Agar Plate (BAP)
Type: Enriched and differential media.
Enriched Purpose: Fortified with 5 ext{%} sheep's blood, providing extra nutrients and growth factors to support the growth of fastidious bacteria. Also known as Chocolate Agar if the blood is heated, lysing red blood cells and releasing nutrients.
Differential Purpose: Differentiates bacteria based on their hemolytic capabilities (ability to lyse or break down red blood cells).
Beta Hemolysis: Complete destruction of red blood cells. This results in a clear, transparent zone around the bacterial colonies where the red color of the blood agar has completely disappeared. It's often possible to see text through the agar in these clear areas.
Alpha Hemolysis: Partial or incomplete breakdown of red blood cells. This produces a greenish discoloration around the colonies due to the conversion of hemoglobin to methemoglobin.
Gamma Hemolysis: No hemolysis (no breakdown) of red blood cells. The bacteria grow on the surface, but the agar directly under and around the colonies remains unchanged in color, appearing red.
Identification: The pattern of hemolysis (beta, alpha, or gamma) helps in identifying specific bacterial species, particularly Streptococcus species.
Inoculation: Streak Plate Method
Definition of Inoculation
Inoculation is the act of introducing microorganisms (bacteria, fungi, etc.) onto a growth medium, such as agar in a Petri dish, to encourage their growth and multiplication.
Streak Plate Method Purpose
The primary purpose of the streak plate method is to thin out a concentrated bacterial sample across the surface of an agar plate until individual, isolated colonies can be observed. This allows for the separation of different bacterial species within a mixed sample, as different species often form colonies with distinct appearances (e.g., shape, color, size).
Materials
Inoculation Loop: A sterile metal wire with a loop at the end, used to pick up and spread bacterial samples.
Bunsen Burner/Electric Heater: Used to sterilize the inoculation loop by heating it to red hot.
Agar Plate: A petri dish containing solid growth media.
Bacterial Sample: The initial culture or specimen (e.g., urine sample) containing bacteria.
Procedure for Streak Plate Method (Four-Quadrant Streaking)
Sterilize the Loop: Heat the entire wire portion of the inoculation loop in a flame until it glows red hot. This kills any contaminants.
Cool the Loop: Allow the loop to cool for a few seconds (without touching any surfaces) to prevent killing the bacterial sample.
Obtain Sample: Dip the cooled, sterile loop into the original bacterial sample (e.g., patient urine).
Quadrant 1 Streaking: Gently lift the lid of the agar plate and streak the loop back and forth across a small area, approximately one-quarter (1/4) of the plate (Quadrant 1). This area will have very heavy bacterial growth.
Re-sterilize and Cool Loop: Re-flame the loop until red hot and let it cool again. Do not re-dip into the original sample.
Quadrant 2 Streaking: Rotate the plate. Touch the cooled loop to the edge of the growth in Quadrant 1 and drag a small amount of bacteria from Quadrant 1 into Quadrant 2. Streak back and forth only within Quadrant 2, making sure to cross into Quadrant 1 only once or twice at the beginning to pick up bacteria. The growth in Quadrant 2 will be less dense.
Re-sterilize and Cool Loop: Repeat step 5.
Quadrant 3 Streaking: Rotate the plate. Touch the cooled loop to the edge of the growth in Quadrant 2 and drag a small amount of bacteria into Quadrant 3. Streak back and forth only within Quadrant 3, crossing into Quadrant 2 only once or twice. Growth in Quadrant 3 will be even more thinned out.
Re-sterilize and Cool Loop: Repeat step 5.
Quadrant 4 Streaking: Rotate the plate. Touch the cooled loop to the edge of the growth in Quadrant 3 and drag a small amount of bacteria into Quadrant 4. Streak back and forth only within Quadrant 4, crossing into Quadrant 3 only once or twice. This quadrant should produce well-isolated colonies.
Final Sterilization: Re-flame the loop one last time to sterilize it before putting it down.
Incubation: Invert the plate and incubate it at an appropriate temperature (usually for approximately 24 hours) to allow bacterial growth.
Common Mistake
The most common error in streak plating is failing to re-sterilize the inoculation loop between each quadrant. If the loop is not flamed, it continues to carry a high concentration of bacteria from the previous quadrant, leading to heavy, un-thinned growth across the entire plate and no isolated colonies.
Bacterial Colony
Definition
A bacterial colony is a visible cluster or mound of bacterial cells growing on the surface of a solid medium. Each colony is typically considered to have originated from a single bacterial cell (or sometimes a small group of cells) that multiplied repeatedly through cell division.
Significance
Observing individual colonies is crucial for microbiology studies because:
It allows for the determination of bacterial species based on unique colony morphology (shape, color, size, texture).
It enables the isolation of pure cultures from a mixed sample, as each distinct colony theoretically represents a pure population of a single species.
Appearance
Different species of bacteria will form colonies with distinct appearances. For example, a mixed sample could show red colonies, clear/tan colonies, and yellow colonies, each representing a different bacterial species.
Culture Types
Pure Culture
A pure culture contains only a single, known species of microorganism. For example, a test tube might contain thousands of individual E. coli cells, but only E. coli (one species) is present. Pure cultures are essential for studying specific microbial characteristics without interference from other organisms.
Mixed Culture
A mixed culture contains two or more identified and easily differentiated species of microorganisms growing together. Although multiple species are present, their identities are known, and they can often be distinguished visually (e.g., by different colony colors on an agar plate) or through further testing.
Contaminated Culture
A contaminated culture is one that was initially intended to be pure or mixed but now contains an unwanted, unidentified microbe. This contamination can arise from various sources, such as improper sterilization of equipment (e.g., an inoculation loop), accidental exposure to environmental microbes (e.g., sneezing on the plate), or improper aseptic technique during transfers. The presence of an unknown organism renders the original culture unreliable for study.