Basic Lab Procedures

Staining and Culture Preparation

Staining
• Procedures meant to give color to the organisms, making
them easier to see under the microscope.
Types of stains
Simple
Use single dye which can either be aqueous (water – based) or alcohol
based. Quick and easy to visualize cell shape, size, and arrangement of
bacteria. Ex. Safranin, methylene blue, crystal violet
Differential
Use to differentiate one group of bacteria from another.
* Gram stain – distinguish gram (+) and (-) bacteria.
All cocci are gram (+) exp. Neisseria, Veilonella and Branbamella
All bacilli are gram (-) exp. Corynebacterium, Clostridium, Bacillus and
Mycobacterium
• Acid – fast stain - use for bacteria with high lipid content in cell wall
Ziehl – Neelsen stain (hot) and Kinyoun stain (cold)
Special
Use to demonstrate specific structures in a bacterial cell.
Ex. Hiss stain (capsule/slime layer), Dyer stain (cell wall), Fischer – Conn
stain (Flagella), Dorner and schaeffer –fulton (spores), India ink/nigrosine
(capsule of the fungus)
ZIEHL – NEELSEN STAIN –
“hot method”
Requires steam – bathing the prepared smear after
addition of the primary dye.
The primary stain used is aqueous and will not bind
to the cell wall of the organisms
Acid – fast organisms will appear red on a blue
background

KINYOUN STAIN
“cold method”
Does not utilize heat after addition of the primary
stain, which is oil – based.
Acid – fast organisms will appear red on a green
background.
Culture Media
An aqueous solution to which all necessary nutrients essential for the growth
of organisms are added. Media (s. medium) are used to grow microorganisms
According to Physical State

Liquid media Semi – solid media Solid media

According to Chemical composition

Synthetic media Non – synthetic media

According to Physical state
• Liquid Media
• Commonly called broths, milk or infusions, these are water –
based solutions that do not solidify at temperatures above the
freezing point. Contain specific amounts of nutrients but do not
contain gelling agents such as gelatin or agar
• Semi – solid Media
• Exhibit clot –like consistency at ordinary room temperature and
contain agar at concentrations of 0.5% or less that allows
thickening of the media without producing a firm substance.
Suited for microaerophilic bacteria or study of motility
• Solid Media
• Contain solidifying agent such as 1.5% - 2% agar, giving them a
firm surface on which cells can form discrete colonies. Used for
isolation of bacteria and fungi or for determining the colony
characteristics of the organism under study.
According to chemical composition
• Synthetic Media
• Contain chemically – defined substances which are
pure organic and / or inorganic compounds. Maybe
simple or complex depending on what supplement is
added to it.
• Non – Synthetic Media
• Complex media, contain atleast 1 ingredient that is
not chemically defined, which means that it is neither
a simple or pure compound
• Most are extracts of animals, plants or yeasts.
Culture Media
According to functional type

General purpose Enrichment Selective

Differential Transport Anaerobe

According to Functional Type
• General Purpose Media – Designed for primary isolation of a
board spectrum of microbes and contain a mixture of nutrients
that support the growth of both pathogenic and non – pathogenic
organisms
ex. Nutrient agar

• Enrichment Media – Contain complex organic substances such as

blood, serum or special growth factors and are designed to
increase the number of desired microorganisms without
stimulating the rest of the bacterial population
2 commonly used enrichment media
• Blood agar – some gram (+) bacteria produce EXOTOXINS that cause
hemolysis
• Chocolate agar – used for the culture of organisms such as Hemophilus
sp. Heat is applied to lyse the red blood cells, causing medium to turn
brown.
According to Functional Type
• Selective Media
• Contain one or more substances that encourage the growth
of only a specific target microorganism and inhibit the
growth of others. Designed to prevent growth of unwanted
contaminating bacteria or commensals so only the target
bacteria will grow.
Ex. Thayer – Martin agar – for isolation of Neisseria, Mannitol
Salt agar – isolation of S. aureus, Lowenstein – Jensen
Medium – for Mycobacterium

• Differential Media
• Allow the growth of several types of microorganisms.
Designed to show visible differences (morphologic
variations) among certain groups of microorganisms
According to Functional type
• Transport Media – Used for clinical specimens that
need to be transported to the laboratory immediately
after collection. Prevent drying of specimen and inhibit
the overgrowth of commensals and contaminating
organisms. Adds charcoal to neutralize inhibitory
factors.
• Anaerobic Media – Media used specifically for
organisms that cannot survive in the presence of
oxygen and require reduced oxidation – reduction
potential and other nutrients. Supplemented with
Vitamin K and hemin. Undergo boiling to remove
dissolved oxygen.
Remember…
Safety First!