Chapter 3: Tools of the Laboratory

The Five I’s of Culturing

1. Inoculation

2. Incubation

3. Isolation

4. Inspection

5. Identification

Inoculation

Addition of microorganism to supporting medium

Inoculation Causative Agents

–Inoculum (sample to be cultured)

–Medium (nutrients allowing growth of inoculum)

–Culture (growth of inoculum in media)

Incubation

Placement of inoculum in conditions to encourage growth

–Temperature

•range between 20 to 45 degrees

–Content of atmospheric gases

•aerobic vs. anaerobic

–Time

•range to 24 hours to several weeks

Physical States of Media

•Liquid

•Semi solid

•Solid

Chemical Composition of Media

•Defined (synthetic) media

•Complex media

Functional Types of Media

•General purpose

•Enriched

•Selective media

•Differential media

•Anaerobic (reducing) media

Liquid Physical State of Media

–Water-based solution

–Growth appears as cloudiness or turbidity

Semi Solid Physical State of Media

-Contains agar that thickens, but does not form a solid

-Used to determine motility

Solid Physical State of Media

• Agar → complex polysaccharide derived from seaweed

• Allows culture media to form solid surfaces

Defined (Synthetic) Chemical Composition of Media

• Exact chemical composition is known

• Used for organisms with strict need for specific growth factors

• Not used for most clinical cultures

Complex Chemical Composition Media

• Exact composition is not quite known

• Contains extracts from yeast, meat, and plants

• Most commonly used

General Purpose Functional Type Media

• Complex media to support growth of as many microbes as possible

• Nutrient broth/agar, trypticase soy agar

• “Anything will really grow in it”

Enriched Functional Type Media

• Contains additional substances required for growth of organism

• Blood agar

• “Same as general purpose media, but blood is added to it”

Selective Media

• Contains substances that favor the growth of particular organisms or inhibit the growth of unwanted ones

1. Mannitol salt agar → allows for the growth of halophiles

2. Sabouraud dextrose agar selectively allows for the growth of fungi and not bacteria

Differential Media

1. Supports growth of many microbes, HOWEVER…

   1. Microbial use of the media or appearance of colonies are different

      1. Helps characterize/identify microbes

   2. Blood agar → differentiates between ability of bacteria to digest red blood cells

   3. Carbohydrate tubes → differentiates between the ability to ferment types of sugars and the resulting products

   4. MacConkey Agar → selective for Gram - bacteria, differentiates between non-lactose and lactose fermenting Gram - bacteria

Anaerobic (Reducing) Media

1. Obligate anaerobes need to be cultured in the absence of oxygen

   1. Agar stab

   2. Reducing media chemically combines with free oxygen and removes it from media (thioglycolic acid)

      1. Ex: clostridium perfringens (gangrene) → infected flesh that turns black, streptococcus pyogenes (necrotizing fasciitis) → flesh eating bacteria located in deep wounds

Transport Media

• Special media to allow for transfer of clinical specimens

• Prevent contamination of other people

• Prevent contamination of sample

• Maintain proper growth conditions for survival

Assay Media

• Used by technologists to test the effectiveness of antimicrobial drugs, antiseptics, disinfectants, and preservatives

• Mueller-Hinton Agar → used to determine antimicrobial sensitivity 

Animal and Cell Special Culture Techniques

• Not all organisms will grow in media

• Need live host cells or host organism

1. Examples:

   1. Chlamydia (STI) and Rickettsia (Rocky Mountain Spotted Fever)

   2. Mycobacterium leprae (leprosy)

   3. Come from armadillos

2. Treponema pallidum (syphilis)

3. Viruses (hot specific cells)

   1. Replicate themselves through host

Mixed Culture

Contains two or more microbes

Pure Culture

• Composed of a single type of microbe

1. Colony consists of millions of offspring from one cell, visible on social media

2. Original cell termed by a colony forming unit (CFU)

Isolation Techniques for Pure Cultures

3 common methods to obtain pure cultures

1. Streak plate

2. Pour Plate

3. Spread Plate

Macroscopic Inspection and Identification

• Colony morphology (size, shape, color)

• Growth in specific media (nutrient and oxygen use, products produced during growth)

• Growth at bottom of test tube media = anaerobic

Microscopic Inspection and Identification

• Size, shape, and staining patterns

• Gram positive or gram negative

• Genetic (genotypic testing for DNA or RNA)

• PCR Testing to determine if strains are similar, toxicity, etc.

• Immunologic (antibody diagnostic tests)

• Ex: Covid-19 test to determine if specific microbe/pathogen is present 

Gram Bacteria

Bacteria classified by the color they turn in the staining method

MacConkey Agar

This substance is used as a selective and differential medium

Blood Agar

This substance is only in a differential medium