Bacterial Staining Techniques & Growth Requirements

Simple staining method

A watery solution of a basic dye is applied to the smear and the organism takes up the color of the dye.
Basic dyes (cationic + charge) will have a strong affinity for the negatively charged cytoplasm of the bacterial cell.

(ex: methylene blue, crystal violet or basic fuschin)

Differential staining

Differential stains are used to distinguish one type of cell from another. Two examples are the Gram stain and the Ziehl Neelson stain.

Differential staining steps

• Application of the first dye or primary stain
• Differentiation: application of a solution that removes the primary stain from some cells
• Counterstain: application of the secondary dye (contrasting color with the primary dye)

Gram stain steps and results

➢ Application of the primary stain: crystal violet

➢ Application of mordant: iodine – potassium – iodide acetone

➢ Critical step – decolorization: alcohol

➢ Counterstain: second dye: safranine

RESULTS:

❑ Gram positive organisms: purple (retain the primary dye)
❑ Gram negative organisms: pink (decolorized by alcohol, they are stained by
safranine

Ziehl Neelsen Stain

This is a differential stain for the tubercle bacilli (Mycobacteria)
Organisms with the ability to retain the primary stain after the decolorization step are term acid-fast.
Organisms that lose the primary stain when treated with acid-alcohol and take the color of the counterstain are termed non acid-fast

Spore stains

▪ Staining is facilitated by application of heat (steaming the preparation)
▪ Procedure: Modified Ziehl Neelsen
• Primary stain: carbol-fuschin and heat
• Decolorization: weak decolorizer 0.25% sulfuric acid solution
• Counterstain: use od a basic dye – malachite green to stain
the cell
▪ Results: spores are pink, cell is green

Physical growth requirements of bacteria

pH (slight alkaline pH, 7.2 to 7.6 ), temperature and atmospheric conditions (require various concentrations of O2 and CO2)

Atmospheric requirements determination method

Thioglycolate broth

Aerotolerance of bacteria

Strict aerobe, Strict anaerobe, Fac. aerobe, Microaerophile, Aerotolerant

Organisms that require carbon dioxide

capnophilic or carboxyphilic.

To increase CO2 in growth conditions we can use a CO2 incubator or a candle jar.

Method to cultivate anaerobes

Anaerobic jar

Control elements of anaerobic jar

Biological indicator (known anaerobic culture plate), redox indicator (methylene blue)

Categorization of bacteria in relation to their temperature requirements.

Psychrophiles (-30- -5), mesophiles (15-45), thermophiles (65-70)

Osmotic pressure

usually refers to the salt concentration of the medium (0.5% NaCl)

Halophilic organisms

10% NaCl concentration is essential for their growth

Haloduric organisms

organism which will tolerate media with a high concentration of NaCl, but which do not require it for growth

photochromogens

produce
pigments in the presence of light.

Autotrophs

microorganisms that use carbon dioxide as their major or sole source of carbon

phototrophs

energy is obtained from sunlight

chemotrophs

energy is obtained by oxidation of inorganic compounds

Heterotrophs

microorganisms that require an organic form of carbon for their growth, i.e. glucose

How energy is obtained by microorganisms

Energy is obtained from oxidation/reduction reactions.

Sources of nitrogen provided in culture media

peptones:
▪ are partially hydrolyzed proteins
▪ do not coagulate by heat and are soluble in water

growth of a culture may be divided into four phases:

Lag phase: cells increase in size

Log phase: active phase of division Cell # increase exponentially

Stationary phase: overall # is constant Bacteria dying =bacteria multiplying Decline phase: number of cells dying exceeds the number reproducing