Micro - Fundamentals of Microbial Growth & decontamination

Bacterial Growth

• asex CD → new daughter cells

• increase total cell pop

• binary fission (when nutr require are met, cell enlarges in size, replicate cell components & eventually divide)

• rate impacted by environ

generation time

• eukaryotic def involving successive cycles of life does not apply

• bacteria can grow rapid or remain dormant for thousands of years

• bacteria def the time it takes for the pop to double through one round of BF

• rates vary enormously (dependent on environ & organism type)

Growth Phase

• bac has 4 district growth phases

• culture density is defined as the number of cells per unit volume

• correlation exist with infection, depending on site & infection type

• 1 = lag phase (adjust to enviorn)

• 2 = log phase (most rapid & active)

• 3 = stationary phase (fill environ with waste; number of cells dividing = number of cells dying)

• 4 = death phase = cells die & waste accumulates & nutrients deplete

Bacteria Adapt to Various Growth Conditions

• enviorn factors impact growth, metab, structure & survival

• survives by adaption to niche conditions: temp, pH, salinity, o2 levels, nutrient availability

Temperature Conditons

• important components

• can’t regulate internal temp & needs membranes/protein to function

• 3 principal temps specific to each:

  • minimum: lowest that support growth

  • optimal: cellular growth highest

  • maximum: highest that support growth

Bacterial classification using temperature

• fundamentally important factor used to classify microbes

• can survive freezing, just not grow until temp favorable

• human pathos (mesophiles) ~ 35-37 C

Bacterial class using pH

• every microbe has max, min & optimum range of pH

• extreme pH affects structure of all macromolecs = evolutionary adaptions

• optimum growth pH is the most favorable for the growth

• acidophiles = grow at pH 1 to pH 5

• neutrophiles = 5-8; majority of microorganisms

• alkaliphiles = 9-11; soda lakes

High salt conditions

• bacterial cyto is 80% water = most cells undergo plasmolysis in high salt

  • h2o rushes out d/t osmosis

• halophiles ~ salt loving

  • tolerate up to 35%, need for growth

  • associated with dead sea & great salt lake

• facultative halophiles

  • dont require high salt but can survive & divide in presence

Oxygen conditions

• many microbes on planet live without or with o2

  • low beneath soil & in silt under lakes/oceans

  • most pathos thrive in low 02

  • anaerobic

• atmospheric molec o2 easily diffuse

  • bacteria varying o2 requirements & tolerances for optimal growth & survival

  • visualized using by growing bacteria in thioglycolate tube culture

  • o2 slowly diffuses throughout thioglycolate tube from top

  • bacterial density increase where o2 concentration is optimal for organisms

  • major category of classification

Bacterial class by o2 use

• obligate aerobes = absolute dependence on o2 for growth & cell process

• obligate anaerobes = do not use o2 may die in presence

• facultative anaerobes = thrive in o2 but can grow without it

• aerotolerant anaerobes = tolerante o2 but neither need or use it

• microaerophiles = live in low o2 need & use in small amounts

reactive o2 species

• however → o2 use for energy creates reactive o2 species

• highly unstable & reactive - can damage virtually macromolecules

  • singlet o2

  • superoxide

  • peroxides

  • hydroxyl radical

• aerobic organisms must be able to detoxify ROS

  • prevent damage to DNA, proteins, lipids → cell death

Detox of ROS

• 3 main enzymes break town toxic byproducts from aerobic: superoxide dismutase, peroxide, catalase

• anaerobes have none; aerobes have all 3, aerotolerant have sod

• basis for useful & rapid test to distinguish 2 major genera of gram pos cocci (strep = aero, staph = facultative)

Catalase Test

• Catalan covert hydrogen peroxide→ water + o2

• isolated colony from streak plate is rapidly mixed into drop of 3% hydr. peroxide

• if the culture is catalase positive ~ released bubbles of o2

Nutrients

• all living things require nutrients, growth factors, & energy source

• microbes use nurtrients from environment to divide, build structural components, macromolecules, enzymes, etc

• essential nutrients:

  • required to replicate, build new cells & maintain/repair

  • found in organic & inorganic compounds of microbes environment

  • macro need in large amount, micro need in small

Growth Factors

• necessary substances for growth/survivial that a cell can’t make on it own

• cells must import required substances from environ

• fastidious = organisms that need multiple growth factors

  • to grow these organisms in lab, AA, vitamins & nitrogenous bases must be supplied

  • intracell

Culture Media

• bacteria is grown in lab using various culture media

• classified by physical state, chemical comp & function

• physical state of media used depends on application

  • liquid = large batches

  • solid = isolated colonies & observing characteristics

  • semisolid = motility test

Complex, enriched & defined media testing

• complex = general all purpose media that support growth of a large variety of bacteria

  • contains extracts & digests of yeasts, meat or plants

  • amount of individual components & specific chem components unknown

  • TSB, NB, BHI

• enriched = complex media + added growth factors, vita, other essential to promote fastidious organisms growth

  • can not make certain nutrients & require additional

• chemically defined = individual chemical components are added in exact amount, complete chemical composite IS known

Selective & Differential Media

• clinical sample = not pure culture

  • urine/fecal, mucosal, biopsy

  • must be able to separate out pathos from normal biota

• selective = inhibit growth of unwanted micro & support growth of organism of interest by supplying specific nutrients & reducing competition

• differential = easy to distinguish by change of color in colonies or medium & color changes are result of end products created by interaction of bacterial enzymes with differential substrates in medium

Selective Media Example

• Neisseria gonorrhoeae & N. meningitidis = fastidious gram neg diplococci

• all neisseria require rich, enriched, supplemental “chocolate media” for lab growth

• use of Ruch media for clinical swab would result in bacterial overgrowth

• selective chocolate media developed to inhibit growth of all others (cocktail of antimicrobials that inhibit gram pos, all other gram neg & fungi)

MacConkey Agar

• selects for growth of gram neg enteric bacteria

• differentiates lactose fermenting from lactose non fermenting gram neg

• used to isolate & identity coliform & intestinal pathos in water, dairy products & bio specimens

Mannitol Salt Agar

• selective for staphylococci (gram pos thrive in salt) inhibits others

• differentiates between staphylococcal species based (only dominant human patho staphylococcus aureus able to ferment mannitol sugar)

Blood Agar differential

• some bacteria species produce & secrete exotoxins

• hemolysins - type of exotoxin that Is extra cell enzyme that lyses rbcs

• hemolysis - can be observed by growing bacteria on complex agar containing animal blood

• produced hemolysis radially diffuse outward from colonies causing complete or partial destruction of the RBC in the medium

• after incubation plate must be held to light source

  • alpha (partial) → green

  • beta (complete) → clear

  • y (none) → no change

Physical & Chemical methods of microbial control

• suffix -cide or -cidal = methods that kill targeted microorganisms

• -stat or -static = do not kill but stop growth

  • less toxic to host

  • keep microbial population in check “stasis”

  • typically sufficient in for healthy individual, prevent patho from multiply

• factors determine if a treatment is cidal or static

Physical methods to control microorganisms

• temp, radiation (damage NA & essential biomolecs), filtration, desiccation (dehydration stasis)

• nonspecifically killed by

  • disrupting membranes or membrane perm

  • damage proteins & NA by denature, degradation or chem modifications

Temperature - cold & heat microbial control

• fridge & freezing slow microbial growth

• heat is most common & oldest form of control (sterile or decontamination)

• moist heat

  • boiling ~ 5 min of rapid boiling eliminated most microbes

  • pasteurization ~ eliminate patho & reduce harmless microbes that spoil

  • autoclaves ~ steam & pressure sterilization

• dry heat (incinerations - flame seconds, hot air oven - hours)

Chemical methods to control Microorganisms

• wide variety of chemical can be used as disinfectants or antiseptics

• germicides = chem that control microbial contamination

  • disinfectants (treat inanimate)

  • antiseptics (living)

  • low level agent (destroy most bac, fungi & some viruses but not endospores)

  • intermediate (destroy all bac & fungi, bu not all vireuses & endos, presence of organics can interfere with effectiveness)

  • high (destroy all microbes & endo ~ sterilizing)