BACTE LAB: CULTURE MEDIA

What does direct methods such as microscopy and nucleic acid detection provide?

provide early information about bacteria and fungi in infection.

Why is microorganism growth usually required?

Microorganism growth is usually required for accurate identification and sensitivity testing.

What does Gram stain provide?

Gram stain gives preliminary information by revealing cellular morphology, arrangement, and structures, but further testing relies on phenotypic, genotypic, or immunologic methods.

Why does culturing the organism remain essential?

Culturing the organism remains essential to provide enough material for accurate identification and sensitivity testing

What are the principles of bacterial cultivation?

To isolate; to determine; to obtain.

What do bacteria require for growth?

Bacteria require various nutrients and growth factors such as gases, water, ions, vitamins, minerals, nitrogen sources, carbon, and energy.

What are examples of nitrogen sources for bacterial growth?

Peptone, casein, meat or plant infusions.

What usually supplies carbon and energy?

Carbon is usually supplied through carbohydrates (e.g., sugars and their derivatives) and proteins.

What does successful cultivation depend on?

Providing essential nutrients and an appropriate environment for growth.

What are the phases/forms of growth media according to the file?

Liquid (broth); solid (agar); biphasic (born lands); semi-solid (small agar concentration, thioglycollate).

What are the two common phases of culture media?

Liquid (broth) and solid (agar).

What happens when nutrients are incorporated into liquid or solid media?

If the media meets bacterial requirements, cells will multiply to visible levels.

What happens in liquid broth when bacteria grow?

Growth produces turbidity that can be seen with the naked eye

What must be done for accurate interpretation of culture media?

The media must be placed in the right environmental conditions.

What are bacteria with demanding requirements called?

Fastidious

What are bacteria with simpler nutritional requirements called?

Non-fastidious.

What is the visual indication of growth in solid media?

Solid colony formation.

What is the visual indication of growth in liquid media?

Turbidity.

What are broth media?

Media in which nutrients are dissolved in water, allowing bacteria to multiply in suspension.

How is growth indicated in broth media?

By a visible change in the broth from clear to cloudy liquid

What causes turbidity in broth?

Light being scattered by bacterial cells.

How many bacteria are needed for turbidity to be seen by the naked eye?

At least 10^6 bacteria/mL.

What may some broths include to indicate growth?

pH indicators

What pH indicator is given as an example in the file?

Phenol red

What happens to pH indicators such as phenol red?

They change color in response to bacterial metabolism

What can the location of growth in broth reveal?

Oxygen requirements.

Where do obligate anaerobes grow in a broth tube?

At the bottom of the broth tube.

Where do aerobes grow in broth?

At the surface.

Where do microaerophiles grow in broth?

Just below the surface where oxygen is lower than atmospheric levels

Where do facultative anaerobes grow in broth?

Throughout the broth.

What are solid media?

Solid media consist of nutrients plus a solidifying agent, most commonly agar.

What is the most common solidifying agent in solid media?

Agar

At around what temperature does agar melt?

95 C

At what temperature does agar solidify?

50°C.

What are the types of solid agar preparations?

Agar plates, agar slants, and agar deeps.

What are agar plates?

Flat surfaces in petri dishes used for isolating colonies

What are agar slants?

Tubes with a slanted agar surface, useful for storage and maintenance of cultures.

What are agar deeps?

Tubes filled to create a deep agar column, used for oxygen gradients or motility.

What does solid media support?

The growth of visible colonies.

What does a colony therefore represent?

A pure culture.

What does a pure colony contain?

Genetically identical bacteria (clone).

Why is selection of colonies important?

It allows accurate identification and characterization of the organism.

What are the four main groups of culture media according to function?

Enrichment, nutritive, selective, and differential media.

What are enrichment media?

Enrichment media supply nutrients that enhance the growth of particular pathogens, especially when these organisms are mixed with other bacteria in a specimen.

What is the purpose of enrichment media?

To give the target organism a growth advantage over others.

What is an example of enrichment media given in the file?

BCYE.

What does BCYE contain?

L-cysteine and other nutrients essential for the growth of Legionella pneumophila.

What is Legionella pneumophila the causative agent of?

Legionnaires’ disease.

What is another form of enrichment media?

Specialized broths

What are specialized broths used for?

To recover organisms present in very low numbers

When are specialized broths particularly useful?

When initial inoculation on solid media does not yield visible colonies.

What are back-up broths (enrichment broths) designed for?

Organisms that need extra nutrients or protection from competing flora.

What does Thioglycollate broth support?

Anaerobes.

What is LIM broth?

Todd Hewitt broth with colistin and nalidixic acid

What is LIM broth used for?

Selective enrichment of group B strep.

What does Gram-negative (GN) broth enhance the recovery of?

Enteric Gram-negative organisms.

What are nutritive media?

Nutritive media provide the essential nutrients to support the growth of most non-fastidious organisms.

Do nutritive media favor one type of organism over another?

No. These do not favor one type of organism over another

What are nutritive media widely used for?

Routine culture

What are examples of nutritive media?

Tryptic Soy Agar, Nutrient agar and Sabouraud’s dextrose agar.

What is Sabouraud’s dextrose agar used for?

fungi

What do nutritive media allow to grow?

A broad spectrum of bacteria and fungi.

How are nutritive media often used?

As a baseline to compare growth on selective or differential media.

What are selective media?

Selective media contain substances that inhibit the growth of certain organisms while allowing others to grow

What are inhibitory agents in selective media?

Dyes, bile salts, alcohols and acids, and antibiotics.

What is an example of selective media containing sheep blood?

Phenylethyl alcohol agar with 5% sheep blood.

What does phenylethyl alcohol agar inhibit?

The growth of aerobic and facultative anaerobic Gram-negative rods

What does phenylethyl alcohol agar permit?

The growth of Gram-positive cocci

Are inhibited organisms completely eliminated by selective media?

No. The inhibited organisms are not completely eliminated; they may still be present in small numbers.

How can improper incubation weaken the selective effect of a medium?

Improper incubation, such as dehydration of the medium, can weaken the selective effect, allowing contaminants to grow.

What are differential media?

Differential media contain ingredients that allow colonies of different bacterial species to be distinguished based on observable differences, such as colony color, appearance, or biochemical activity.

What observable differences can differential media show?

olony color, appearance, or biochemical activity.

What is an example of differential media?

MacConkey agar (MAC)

What is the selective function of MacConkey agar?

It inhibits the growth of Gram-positive organisms

What is the differential function of MacConkey agar?

It separates Gram-negative bacteria into lactose fermenters and non-lactose fermenters.

What color are lactose-fermenting colonies on MacConkey agar?

Pink to red.

What color are non-lactose-fermenting colonies on MacConkey agar?

Colorless colonies.

What is an example of differential media that uses hemolysis patterns?

Sheep blood agar.

What does sheep blood agar support nutritionally?

Growth of most non-fastidious organisms.

What does sheep blood agar allow differentially?

Observation of hemolysis patterns.

What are the hemolysis patterns observed on sheep blood agar?

Alpha, beta, and gamma.

What is the use of Blood Agar (BA)?

One of the most used media for routine bacteriology. Supports the growth of nearly all clinically significant bacteria except the most fastidious organisms.

What is the composition of Blood Agar?

Protein source (tryptone, soybean protein digest), sodium chloride, agar, and 5% sheep blood.

What blood may be substituted for sheep blood for specific organisms?

Rabbit or horse blood.

What organism is given as an example for blood substitution?

Haemophilus.

What key concept does Blood Agar enable?

Observation of hemolytic patterns caused by bacterial enzymes that lyse red blood cells.

What is beta-hemolysis?

Complete clearing around colonies

What is alpha-hemolysis?

Partial lysis with a greenish discoloration.

What is gamma-hemolysis?

No effect on RBC.

What is the use of Brain-Heart Infusion (BHI)?

A rich medium for fastidious and non-fastidious microorganisms.

How can Brain-Heart Infusion be prepared?

As broth or agar, with or without added blood.

What is the composition of Brain-Heart Infusion?

Infusions of animal tissue (brain, heart), peptones (protein source), phosphate buffer, and dextrose.

What is Brain-Heart Infusion suitable for?

Antimicrobial susceptibility testing and establishing bacterial identification.

What is Chocolate Agar used for?

An enriched medium for fastidious organisms that cannot grow on BAP.

How is Chocolate Agar composed?

Same as BA, but blood is added to molten agar, lysing RBC.

What does lysing RBC release in Chocolate Agar?

Heme (X factor) and NAD (V factor)

Which bacteria require the X and V factors?

Bacteria like H. influenzae and N. gonorrhoeae.

What is the appearance of Chocolate Agar?

Brown color due to lysed red cells, hence the name “chocolate agar.”