Controlling Microbial Growth

In Vitro

Chapter 8

Factors that Affects Bacterial Growth

• Availability of nutrients

• Moisture

• Temperature

• pH

• Osmotic pressure and salinity

• Barometric pressure

• Gaseous atmosphere

Availability of Nutrients

• All living organisms require nutrients to sustain life.

• To survive, appropriate nutrients must be available.

• Catabolism and anabolism

• Essential nutrients, elements and trace elements

• About 25 of the 92 naturally occurring elements are essential to life.

Moisture

• Water is essential to life.

• Cells consist of 70-95% water.

• Water is required to carry out normal metabolic processes.

• Endospores and cysts can survive complete drying process (desiccation).

Temperature

• Optimum growth temperature

• Minimum growth temperature

• Maximum growth temperature

• Thermophiles

• Mesophiles

• Psychrophiles– Psychrotrophs- refrigerator temperature– Psychroduric organism- can endure

freezing temperature

pH

• Acidity or alkalinity• Most microorganism prefer a neutral or

slightly alkaline medium (pH 7-7.4)• Most bacteria grow between pH 6.5 and 7.5• Molds and yeasts grow between pH 5 and 6• Acidophiles• Alkaliphiles• Vibrio cholerae is the only human pathogen

that grows well above pH 8.

Osmotic Pressure and Salinity• Osmotic pressure- pressure that is exerted on a

cell membrane by solutions both inside and outside the cell.

• Osmosis• Hypertonic• Crenation • Plasmolysis • Desiccation• Hypotonic• Hemolysis• Plasmoptysis• Isotonic• Halophilic and haloduric organisms

Plasmolysis

Barometric Pressure

• Most bacteria are not affected by minor changes in barometric pressure.

• Some thrive at normal atmospheric pressure (about 14.7 psi).

• Barophiles- thrive deep in the ocean and in oil wells, where the atmospheric pressure is high.

Gaseous Atmosphere

• Oxygen (O2)

Obligate aerobes

Facultative anaerobes

Obligate anaerobes

Aerotolerant anaerobes Microaerophiles

Encouraging the Growth of Microorganism In Vitro

• Gather information in the identification of any pathogens present.

• Learn more about microorganisms.

• Harvest antibiotics and other microbial products.

• Test new antimicrobial agents and produce vaccines.

• Viruses, bacteria, fungi and protozoa, with emphasis on bacteria.

Culturing Bacteria in the Laboratory

• petri dishes

• test tubes

• bunsen burners/alcohol lamps

• wire inoculating loops

• bottles of staining reagents

• incubators

Bacterial Growth

Microbial growth = increase in number of cells, not cell size

Generation Time

• The time it takes for one cell to become two cells by binary fission.– Rapid growers (short GT)– Slow growers (long GT)

• E. coli, V. cholerae, Staphylococcus and Streptococcus- 20 mins.

• Pseudomonas and Clostridium- 10 mins.• M. tuberculosis- 18 to 24 hours

Culturing Bacteria

• Fastidious- with complex nutritional requirements

• Using culture media

• Obligate intracellular parasites- do not grow in culture media

• Treponema pallidum and Mycobacterium leprae

Culture Media

• Artificial media or synthetic media- they are prepared in the laboratory

• Culture medium- nutrients prepared for microbial growth

• Inoculation- introduction of microbes into medium

• Culture- microbes growing in/on culture medium

Classification of Culture Media Based on Whether the Exact Contents are Known

• Chemically defined media- exact chemical composition is known

• Complex media- exact contents are not known, from extracts and digests of yeasts, meat, or plants

Liquid and Solid Media

• Liquid media- or broths are contained in tubes, referred to as tubed media.

• Solid media- prepared by adding agar to liquid media and then poured into test tubes or petri dishes, where the media solidifies.– Agar plate– Agar slant– Agar butt/deep

Enriched Medium

• Broth or solid medium containing rich supply of special nutrients that promotes the growth of fastidious organisms.

• Prepared by adding extra nutrients to a medium called nutrient agar.

• Blood agar and chocolate agar

• N. gonorrhoeae and H. influenzae

Selective Medium

• Has added inhibitors that discourage the growth of certain organisms without inhibiting growth of the organism being sought.

• MacConkey agar- inhibit growth of Gram (+) bacteria and is selective for Gram (-) bacteria.

• Phenylethyl alcohol agar (PEA) and colistin-nalidixic acid agar (CNA)- inhibit growth of Gram (-) bacteria.

• Thayer-Martin agar and Martin-Lewis agar- selective for N. gonorrhoeae.

• Mannitol salt agar (MSA)- only for salt-tolerant (haloduric) bacteria

Differential Medium

• Permits the differentiation of organisms that grow on the medium.

• MacConkey agar- used to differentiate various Gram (-) bacilli that are isolated from fecal spcimens.– Gram (-) bacteria are able to ferment lactose

produces pink colonies, those are unable to ferment lactose produce colorless colonies.

– Differentiates between LF and NLF Gram (-) bacteria.

• Mannitol salt agar- used to screen for S. aureus, pink to yellow.

Remember…

• Various categories of media are not mutually exclusive.

• Ex: blood agar is enriched and differential• MacConkey agar and MSA are selective and

differential• PEA and CNA are enriched and selective• Thayer-Martin and Martin-Lewis are highly

enriched and highly selective• Thioglycollate broth (THIO) is a liquid

medium that supports the growth of all categories of bacteria.

In the history…

• Robert Koch- described his culture techniques in 1881.

• Fanny/Frau Hesse- suggested the use of agar.

• Richard Julius Petri- invented the glass Petri dishes.

• Joseph Lister- the first person to obtain a pre culture of bacterium (Streptococcus lactis) in a liquid medium.

Inoculation of Culture Media

• Inoculation- adding a portion of the specimen to the medium.

• Inoculaton of a solid or plated medium involves the use of sterile inoculating loop to apply a portion of the specimen to the surface of the medium; a process commonly referred to as “streaking”.

Streaking the Agar Plate

Importance of Using “Sterile Technique”

• Necessary to exclude all microorganisms from a particular area, so that area will be sterile.

• Media should remain sterile before inoculation.

• Contaminants- unwanted microorganisms• Contaminated- if the sample contains

contaminants

Incubation

• After media are inoculated, they must be incubated, and placed in a chamber (incubator).

• To culture most human pathogens, the incubator is set at 35 – 37 o C

• Carbon dioxide incubator – 5 to 10%, is used to isolate capnophiles

• Non-carbon dioxide incubator – 20 to 21 % of Oxygen

• Anaerobic incubator

Bacterial Population Counts

• Determine the total number of bacterial cells in the liquid

• Determine the number of viable cells

• Spectrophotometer

• Viable plate count– Is used to determine the number of viable

bacteria in a liquid sample such as milk, water, ground food diluted in water, or broth culture.

Spectrophotometer

• Turbidity

Viable Plate Count

• Number of colonies must be multiplied by the dilution factors.

• If 220 colonies were counted on the agar plate that had been diluted with a 1.0-ml sample of a 1:10,000 dilution, there were:

• 220 X 10,000= 2,200,000 bacteria/ml

Viable Plate Count

• Plate Counts: Perform serial dilutions of a sample

Viable Plate Count

• Inoculate Petri plates from serial dilutions

Viable Plate Count

• After incubation, count colonies on plates that have 25-250 colonies.

Bacterial Population Growth Curve

• Determined by growing a pure culture of the organism in a liquid medium at a constant temperature.

• Data are plotted on a graphic paper, plotting the logarithm (log10) of the number of viable bacteria (y-axis) against the incubation time (x-axis).

Bacteria Population Growth Curve

Phases of the Growth Curve

• Lag phase- during which the bacteria absorb nutrients, synthesize enzymes, and prepare for cell division, the bacteria do not increase in number.

• Log phase- exponential growth phase; bacteria multiply so rapidly that the number of organisms double with each generation time.

• Stationary phase- the number of bacteria that are dividing equals the number that are dying; greatest population density.

• Death/decline phase- culture may die completely

Bacteria Population Growth Curve

Culturing Obligate Intracellular Pathogens in the Laboratory

Culturing Fungi in the Laboratory

• Brain Heart Infusion Agar

• Sabouraud Dextrose Agar- pH 6.5 selective for fungi

Culturing Protozoa in the Laboratory

• Acanthamoeba spp.• Entamoeba hisolytica• Balamuthia spp.• Giardia lamblia• Leishmania spp.• Trypanosoma cruzi• Toxoplasma gondii• Trichomonas vaginalis• Naegleria fowleri

Inhibiting the Growth of Microorganism In Vitro

• Sterilization– Dry heat

– Autoclaving (steam under pressure)

– Gas (ex. ethylene glycol)

– Various chemicals (formaldehyde)

– Radiation (UV, gamma rays)

Disinfection, Pasteurization, Disinfectants, and Sanitization

• Disinfection- removal of pathogens from nonliving objects by physical or chemical methods. Ex. Pasteurization

• Disinfectants- are strong chemical substances that cannot be used on living tissue.

• Antisepsis- removal of pathogens from living tissue

• Sanitization- lower microbial counts on eating utensils

Microbial Agents

• Biocidal agents/ Germicidal agents/ Microbicidal agents- are disinfectants that kill microbes

• Bactericidal agents- disinfectants that specifically kill bacteria but not necessarily bacterial endospores.

• Sporicidal agents- to kill bacterial endospores• Fungicidal agents- to kill fungi, including fungal

spores• Algicidal agents- to kill algae in swimming pools and

hot tubs.• Viricidal agents- destroy viruses• Pseudomonicidal agents- Pseudomonas species• Tuberculocidal agents- kill M. tuberculosis

Microbistatic Agents

• Microbistatic agent- is drug or chemical that inhibits growth and reproduction of microorganism

• Bacteriostatic agents- is one that specifically inhibits the metabolism and reproduction of bacteria.

• Lyophilization- is a process that combines dehydration and freezing.– To preserve foods, antibiotics, anti-sera,

microorganisms

Sepsis, Asepsis, Aseptic Technique, Antisepsis, and Antiseptic Technique

• Sepsis- refers to microbial contamination or presence of pathogens in blood or tissues

• Asepsis- is the absence of significant contamination.

• Aseptic techniques- prevent microbial contamination of wounds.– Hand washing, use of sterile gloves, masks, and

gowns.– Antisepsis : prevention of infection– Antiseptic Technique- developed by Joseph

Lister, refers to use of antiseptics

• Alternation of membrane permeability

• Damage to proteins

• Damage to nucleic acids

Actions of Microbial Control Agents

• Heat– Temperature and time– Thermal death point (TDP)- lowest temperature at

which all cells in a culture are killed in 10 min.– Thermal death time (TDT)- time to kill all cells in a

culture– Decimal reduction time (DRT)- Minutes to kill 90%

of a population at a given temperature– Dry Heat- Oven, 160 to 165 C for 2 hours or 170 to

180 C for 1 hour.– Incineration- or burning of contaminated

disposable materials

Using Physical Methods to Inhibit Microbial Growth

• Moist heat- denatures proteins

• Autoclave: – Large pressure

cooker– Steam under

pressure– 15 psi, 121.5C,

20 minutes

• Cold

• Low temperature inhibits microbial growth– Refrigeration– Slow freezing– Rapid freezing (liquid N)– Lyophilization (freeze drying)

• Desiccation– prevents metabolism

Using Physical Methods

to Inhibit Microbial Growth

• Radiation- damages DNA– Ionizing radiation (X rays, gamma rays,

electron beams) – Non-ionizing radiation (UV) – Ultrasonic waves– Microwaves kill by heat; not especially

antimicrobial

• Filtration

• Gaseous Atmosphere– altering the atmosphere in which the

microorganisms are located– Ex. Gas gangerene

• Chemical disinfection refers to the use of chemical agents to inhibit the growth of pathogens, either temporary or permanent.

Using Chemical Agents to Inhibit Microbial Growth

Factors to Consider Whenever a Disinfectant is Used

• prior cleaning

• organic load

• bioburden

• contration of disinfectant

• contact time

• physical nature of the object

• temperature and pH

Characteristics of an Ideal Microbial Agent

• broad anti-microbial spectrum• fast acting (short contact time)• not affected by the presence of organic matter• non-toxic and non-corrosive• leave a residual microbial film• soluble in water and easy to apply• inexpensive and easy to prepare• stable, can be stored for long periods• odorless

How do disinfectant kill microorganisms?

• target and destroy cell membranes (triclosan, detergents, alcohols, chlorhexidine and phenolic compounds)

• destroy enzyme and structural enzymes (hydrogen peroxides, formaldehyde, salt of heavy metals, formaldehyde and ethylene oxide)

• attack cell wall or nucleic acids

• Evaluating a disinfectant– Use-dilution test

1. Metal rings dipped in test bacteria are dried

2. Dried cultures placed in disinfectant for 10 min at 20°C

3. Rings transferred to culture media to determine whether

bacteria survived treatment

Using Chemical Agents to Inhibit Microbial Growth

Using Chemical Agents to

Inhibit Microbial Growth • Evaluating a disinfectant

• Disk-diffusion method

Chemical Food Preservatives

• Chemical Food Preservatives– Organic Acids

• Inhibit metabolism• Sorbic acid, benzoic acid, calcium propionate• Control molds and bacteria in foods and

cosmetics

• Nitrite prevents endospore germination

• Antibiotics- nisin and natamycin prevent spoilage of cheese

Microbial Characteristics and Microbial Control

FINISHED…

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