Staining and Bacterial Growth

Staining and Bacterial Growth

staining

• coloring of microorganisms with dye to emphasize certain structures

stains

salts composed of a positive and negative ion, one of which is colored and is known as a chromophore

chromophore

colored ion in stains

basic dye

the color is in cation

acidic dye

the color is in anion

negative staining

• preparing colorless bacteria against a colored background

• valuable for observing cell shapes, size, and capsule

• halo/capsule can be seen

• cells are made highly visible against a contrasting dark background

simple staining

• aqueous or alcohol solution of a single basic dye

• highlights entire microorganism so the cellular shape and basic structure of cells are visible 

mordant

• additive used to increase affinity of stain

• coat a structure to make it thicker and easier to see

examples of mordant additive

• methylene blue - blue in color

• carbolfuchsin - red in color

• crystal violet - violet/purple in color

• safranin - pinkish-red in color

differential stain

• reacts with different kinds of bacteria 

• gram stain and acid-fast stain

gram stain

• developed by Hans Christian Gram in 1884

• most useful staining procedure 

• classifies bacteria into 2 large groups: gram-positive and gram-negative 

• gram reaction can provide valuable information for the treatment of disease

• gram-positive = can be killed by penicillin and cephalosporin

• gram-negative = generally more resistant due to the lipopolysaccharide layer

process of gram staining

1. cover the heat-fixed smear with basic purple dye (crystal violet), referred to as primary stain

2. wash off the purple dye and the smear is covered with iodine, a mordant

3. wash the slide with alcohol or an alcohol-acetone solution (decolorizing agent) which removes the purple from the cells of some species 

4. the alcohol is rinsed off then stained with safranin, a basic red dye

5. the smear is washed, blotted dry, and examined microscopically

acid-fast stain

• binds strongly only to bacteria that have waxy material in their cell wall

• used to identify all bacteria in genus Mycobacterium (M. tuberculosis and M. leprae) and pathogenic strains of Nocardia

process of acid-fast staining

1. red dye carbolfuchsin is applied to a fixed smear and the slide is gently heated for several minutes 

2. slide is cooled and washed with water

3. the smear is treated with acid-alcohol (decolorizer) which removed the red stain from bacteria that are not acid-fast 

4. smear is then stained with methylene blue (counter stain) 

special stains

used to color parts of the microorganism (endospores flagella, or capsules) 

negative staining for capsules

• capsule - gelatinous covering of many microorganisms

• presence of capsule = determining the organism’s virulence (degree to which a pathogen can cause disease)

• more difficult because capsular materials are water soluble

• capsules do not accept most biological dyes such as safranin = halo

process of staining for capsules

1. mix the bacteria in a solution containing fine colloidal suspension of colored particles (India ink or nigrosine) for contrasting background

2. stain the bacteria with simple stains such as safranin

endospore staining

• endospore - a special resistant, dormant structure formed within a cell that protects a bacterium from adverse environmental conditions

• endospores cannot be stained by ordinary methods because they don’t penetrate the endospore’s wall 

• endospores appear green within red or pink cells

Schaeffer-Fulton endospore stain

most commonly used for endospore staining

process of endospore staining

1. malachite green is applied to a heat-fixed smear and heated to steaming for 5 minutes

2. the preparation is washed for about 30 seconds with water 

3. safranin is applied to the smear stain proportions of the cell other than endospores 

flagella staining

• flagella - structures for locomotion too small to be seen

• uses mordant and the stain carbolfuchsin to build up the diameters of the flagella

requirements for bacterial growth

physical

• temperature

• pH

• osmotic pressure

chemical 

• carbon

• nitrogen

• sulfur

• phosphorus

• oxygen 

Temperature

certain bacteria are capable of growing at extremes of temperature that would certainly hinder the survival of almost all eukaryotic organisms

psychrophiles

• cold-loving microbes

• organisms capable of growing at 0 degrees Celsius

• most of these organisms are so sensitive to higher temperature that they cannot grow in a warm room (25 degrees)

• found mostly in the ocean’s depth or polar regions (seldom problems in food preservation)

psychrotrophs

• organisms that can grow at 0 degrees but optimum growth temp is 20-30 degrees and cannot grow above 40 degrees

• mostly encountered in low-temperature food spoilage because they can grow at refrigerator temperatures

• spoilage organisms

mesophiles

• moderate-temperature-loving microbes)

• the most common type of microbe

• optimum temperature for any pathogenic bacteria is about 37 degrees

• includes most of the common spoilage and disease organism

thermophiles

• heat-loving microbes 

• microorganisms capable of growth at high temperatures

• optimum growth: 50-60 degrees

• not considered a public health concern

• important in organic compost piles 

hyperthermophiles or extreme thermophiles

microbes that have an optimum growth temperature of 80 degrees

pH

• most bacteria grow best in a narrow pH range near neutrality (between 6.5 and 7.5)

• very few bacteria grow at an acidic pH below about pH4

acidophiles

microorganisms that are tolerant of acidity

osmotic pressure

• most organisms must be grown in a medium that is nearly all water (1.5% concentration of agar) 

• if osmotic pressure is low (environment is hypotonic), microbes with weak cell wall may be lysed

halophiles

can adapt to high salt concentrations

chemical requirement

• carbon

• trace elements (iron, copper, molybdenum, and zinc)

• oxygen

  • obligate aerobes

  • facultative anaerobes

  • aerotolerant anaerobes

  • microaerophiles 

obligate aerobes

organisms that require oxygen to live

facultative anaerobes

can use oxygen when it is present but are able to continue growth by using fermentation or anaerobic respiration when oxygen is not available

• ex. E.coli

anaerobes

bacteria that are unable to use molecular oxygen for energy yielding reactions

• ex. Clostridium

aerotolerant anaerobes

fermentative and cannot use oxygen for growth, but they tolerate it fairly well

microaerophiles

aerobic; they do not require oxygen

• they grow inly in oxygen concentrations lower than those in air

bacterial growth

• refers to an increase in bacterial numbers, not an increase in the size of individual cells

• bacteria reproduce the binary fission

• few bacterial reproduce by budding

budding

forming a small initial outgrowth that enlarges until its size approaches that of the parent cell, then it separates 

binary fission

• asexual reproduction

• cell elongates and DNA is replicated

• plasma membrane begins to constrict and a new wall is made

• cross-wall forms, completely separating the two DNA copies

• cells separate 

generation time

• time required for a cell to divide

• varies considerably among organisms and with environmental conditions such as temperature

• uses logarithmic scales

phases of growth

1. lag phase

2. log phase

3. stationary phase

4. death phase

lag phase

• preparing for population growth, but no increase in population 

• a period of little to no cell division

• can last for 1 hour or several days

• cells are not dormant

• period of intense metabolic activity such as synthesis of enzymes

• incubation period

log phase

• logarithmic or exponential increase in population

• cell begins to divide and enter a period of growth or logarithmic increase

• cellular reproduction is most active

• generation time reaches a constant minimum 

stationary phase

• period of equilibrium

• microbial deaths balance population of new cells

• growth rate slows

• population stabilizes

• the population exceeds the carrying capacity (number of organisms that an environment can support) and run out of nutrient and space

death phase/logarithmic decline phase

• population is decreasing at a logarithmic rate

• the number of deaths exceeds the number of new cells being formed

• the population is diminished to a tiny fraction of the number of cells or until the population dies out entirely

direct measurement of bacterial growth 

• serial dilution

• counting bacteria by filtration

• Petroff-Hausser cell counter 

• Spectrophotometer