How to Feed and Nurture Your Microbe

cultivation =

culture = “grow”

need to “amplify” to detect, isolate, and characterize microbe

viruses

are filterable infectious agents

virus particles can be enumerated (titer) through serial dilution and infection of tissue culture cells or chicken embryonated eggs

tissue culture(s)

Embryonated chicken eggs can be used to isolate and cultivate animals viruses

Involves lab growth of primary (tissue explant) or immortalized cell lines

These cells require growth factors, glucose, and essential amino acids and vitamins for growth.

Antibiotics limit bacterial contamination of media.

Cell death can be visualized by observing changes in morphology, cell detachment from plastic or staining with trypan blue

complex medium

→ glucose, peptone, & yeast extract

→ supplement: liver extract, fetal calf serum, dead bacteria

protozoans cultivation in labs

some can be cultivated using complex media, and in some cases, bacteria

pathogenic protozoans

some require tissue culture cells for growth while others require some animal host

intestinal parasites

Can be isolated using flotation

Centrifugation in a solution with a high specific gravity

Protozoan cysts, coccidian oocytes, microsporidian spores and certain helminth eggs and larvae are present on the top layer following centrifugation.

the microscope

is an essential tool for assessing the isolation and cultivation of protozoans

cardinal temperatures

growth temperature minimum, optimum, and maximum

heat-shock and cold-shock → “stress” response

optimum = 37 C

stenothermal

small temperature range for growth

ex: Neisseria sp.

Eurythermal

wide temperature range for growth

ex: E. Faecalis

optimal temperature

psychrophiles→ 0-15 C

Mesophiles→ 20-45 C

Thermophiles → >55 C

Listeria→ 4C, cold enrichment

Pasteurization→ 63 C for 30 mins

acidophiles

optimal growth between pH 1-5.5

neutrophiles

optimal growth between pH 5.5 and 8, preferably 6.5-7.5

alkalophiles

optimal growth between pH 8.5 to 11.5

internal pH of all cells

maintained close to neutrality (pH 7)

pathogens/symbionts pH

pH 6.5-7.5

Acid Adaptation (Acid Tolerance Response)

preadaptation phase→ cells exposed to pH 5-6

acid survival: pH <4

proton-translocating ATPase maintains homeostasis (pH 7)

regulated by alternate factors (starvation response)

detoxifying oxygen products

superoxide dismutase

catalase

catabolism

oxidation of organic/inorganic compounds

oxygen or “other” serves as an electron acceptor

does oxygen factor in organism’s metabolism (YES vs NO)

YES:

→ respiration

→ oxygen is a terminal electron acceptor to regenerate NAD+

→ end product: CO2

NO:

→ fermentation

→end product: acids or alcohols

Facultative Anaerobes:

→ if oxygen is present, this pathway is preferred

→ 1 glucose = 36 ATPs

Fermentation

an O2 independent process

problem: catabolism involves the oxidation of organic compounds, which requires NAD+ as an oxidizing agent (NAD+ → NADH + H)

limited amount of NAD+ in bacterial cell

if NAD+ is not regenerated, then catabolism and growth ceases

solution: re-oxidize NAD+ through:

→ electron transport with O2 as terminal electron acceptor

→fermentation

bacterial growth goes through 5 phases

→ lag

→ exponential growth

→ stationary phase

→ death

exponential phase

bacterial population double at a constant rate

This rate of change is dictated by the growth medium/conditions and the organism’s genetics

stationary phase

where the growth rate slows/stops, is the result of the depletion of some essential nutrient or buildup of some metabolic byproduct (ex: acid like lactate)

medium/media

nutrients that promote and support microbial growth

agar: solid medium

Supports microbial growth

Generally made by adding agar, harvested from algae, to some medium

Plates

molten agar poured into a sterile petri dish and allowed to solidify at room temp.

broth: liquid medium

origins come from days when meat broths (soup) were used to grow bacteria

isolation

streaking for isolation: dilute bacteria across the agar surface

dilution to extinction

diluent (buffer or medium)

enumerated bacteria

done through direct microscopic counts, measuring absorbance, or a combination of dilution to extinction with plate counts or growth in liquid culture (MPN)

tergitol

inhibits Gram-positives, molds, and numerous gram-negatives, including Proteus, Providencia, and Pseudomonas species

bacterial properties can be exploited to select or enrich for certain bacteria

→ Crystal/bile salts in MacConkey agar select for gram-neg “enterics” like E. coli and salmonella

→phenyl ethyl alcohol favors gram-pos

→ endospores are resistant to killing by chloroform

→ finally, certain media components or growth conditions favor the growth of certain bacteria over others

cultivation for phages

→ grow bacteria to early exponential phase

→ infect bacteria with phage at low multiplicity of infection

→ grow overnight

→ add organic solvent (chloroform) to release intracellular phage

→ low-speed centrifugation and filtration to remove bacteria and purify phage

assay for phages

phages are viruses of bacteria

bacteria: “food” for phages

plaque assay

visualize viral particles with electron microscope

phages

→ can be isolated and propagated in bacteria

→ requires actively growing bacterial cells (exponential phase), divalent cations, and low MOI. Phages can be released from infected bacterial cells with chloroform

→ phages can be enumerated (titer) by diluting the viral lysate, mixing with growing bacterial cells, and pouring plating bacteria-virus mix onto an agar surface. Phage lyse bacteria produce plaque

growth and care for fungus

→ preferred growth temp for most molds is 25 C (room temp), but some will grow at 37 C

→ not difficult to grow. grow on many complex medium commonly used to culture bacteria and some, odd specialized media

→ because of their ability to form exospores, they are common contaminant in labs (“dust”)

→ are the easiest to grow

→ Molds grow best at room temp.

→ because molds produce “hearty” exospores, they are commonly found on surfaces and can become the bane for many labs

macroelements

nutrients required in large amounts for growth

→ C, O, H, N, S, and P (gram quantities/liter)

→ K, Ca, Mg, and Fe (cofactors)(mg amounts/liter)

microelements

→ trace elements

→ nutrients required in small amounts for growth

→ exist as contaminants in water and glassware. The concentration present in the water is sufficient for growth

→ Mn, Zn, Co, Ni, and Cu (ug amounts/liter)

requirements for C, H (e^-), and O

energy source (catabolism):

→ oxidation of organic/inorganic compounds

→ chemical energy (ATP)

→ energy (ATP) is required for cell growth, motility, “homeostasis”, and transport

Carbon Source (anabolism):

→ biosynthesis: proteins, nucleic acids, polysaccharides and lipids

→reduction

→ reducing power (e-): NADPH

Nitrogen Sources

amino acids

NH₄

NO₃ (nitrate reduction)

N₂ (atmospheric): nitrogen fixation (ex: rhizobium, cyanobacteria)

phosphate sources

inorganic phosphate

sulfur sources

sulfur

growth factors

organic compounds essential for cell growth that cannot be synthesized by the organism

types: amino acids (proteins), purines/pyrimidines (nucleic acid), vitamins (enzymes)

fastidious: having many special nutritional needs that are difficult to meet in the lab (auxotrophy)

pathogens or symbionts are chemotrophs ..

their requirement from some organic molecule (ex: sugar) for growth

it must use this molecule to extract chemical energy as well as make the building blocks for cell growth and division

adequate incubation conditions

temperature

atmosphere (O2, H2, N2, CO2)

growth medium

→ chemically defined medium: nutrients composition and concentration are known

→ minimal medium: minimum amount of nutrients to grow the organism

→ complex medium: composed of relatively undefined components

peptide and amino acid sources (peptones) are not created equal …

→ amino acid content differs: meat, casein (milk protein), gelatin, soybean, yeast, and grains

→ peptides and amino acids are produced from protein by enzymatic (trypsin, chymotrypsin, papain) or acid hydrolysis

→ peptone composition will differ with different enzymes

→ acid hydrolysis destroys tryptophan, cystine, serine, and threonine are decreased, asparagine and glutamine are converted to their acidic forms

sources of amino-nitrogen used in media

yeast extract, pancreatic digest of casein, peptic digest of animal tissue, protease peptone #3, liver digest, beef extract, and peptone (tryptone)

to isolate and grow microbes in a lab, it requires providing it with what?

a carbon and energy source (sugar or amino acid)

salt buffer to maintain neutral pH during growth

some nitrogen source (NH3Cl, peptone, etc)

and a growth factor if the microbe is fastidious

aerobic

normal air to 5-10% CO2

→ CO2 benefits Pasteurella, avibacterium, Neisseria, Streptococcus, and Hemophilus

→ not inhibitory for facultative anaerobes

microaerophlic

85% N2, 10% CO2, 5% O2

→ jars, BioBags, incubators- campylobacter

cannot handle toxic oxygen by-products

anerobic

100% N2 or 100% CO2

candle jars, BioBags, incubators, clove boxes

cannot handle toxic oxygen by-products

temperature

4 C→ can use refrigeration for enrichment of Listeria cultures

37 C→ conventional bacteriology

30 C→ non-fermenting gram neg rods and fungi

42-45 C→ thermotolerant organisms (salmonella, campylobacter, and some yeasts- use temp to control contaminants)

growth rate and yield are influenced by what?

media composition, atmosphere, or temperature

campylobacter culture (1977)

grows at 37 C and 42 C but not at 25C

enriched medium

requires microaerophilic conditions and humid atmosphere (5% O2, 10% CO2, 85% N2)

nonfermentable metabolism (doesn’t produce acid)

old, stressed, damaged cells may become coccoid and not form colonies= viable by nonculturable

can adapt strains to less complex media and ambient atmosphere but they lose virulence and ability to colonize birds

campylobacter nutrition

carbon sources for growth

→ doesn’t use simple carbohydrates such as glucose, lactose, maltose, sucrose

→ poultry isolates can use fucose acquired from chicken mucin

→ utilize the amino acids serine, aspartate, glutamate, asparagine, and glutamine

Auxotrophy (essential amino acids)

→ Methionine

→ some isolates need additional amino acids

anaerobic environment is critical

→ deplete starting material of oxygen (oxyrase)

→ make medium/environment anaerobic (gas mix, oxyrase)

→ agar is “too toxic” (H2O2): replace with gellan-gum or add catalase

media supplements

→ vitamins & amino acids (auxotrophy)

→ siderophores

→ quorum-sensing molecules