Controlling Microbial Growth in Vitro

Controlling Microbial Growth In Vitro : Controlling Microbial Growth In Vitro Chapter 8

Factors that Affects Bacterial Growth : Factors that Affects Bacterial Growth Availability of nutrients Moisture Temperature pH Osmotic pressure and salinity Barometric pressure Gaseous atmosphere

Availability of Nutrients : 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 : 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 : Temperature Optimum growth temperature Minimum growth temperature Maximum growth temperature Thermophiles Mesophiles Psychrophiles Psychrotrophs- refrigerator temperature Psychroduric organism- can endure freezing temperature

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pH : 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 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 : Plasmolysis

Barometric Pressure : 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 : Gaseous Atmosphere Oxygen (O2)

Encouraging the Growth of Microorganism In Vitro : 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 : Culturing Bacteria in the Laboratory petri dishes test tubes bunsen burners/alcohol lamps wire inoculating loops bottles of staining reagents incubators

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

Generation Time : 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 : 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 : 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 : 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 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 : 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 : 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 : 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… : 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… : 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 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 : Streaking the Agar Plate

Importance of Using “Sterile Technique” : 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 : 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 : 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 : Spectrophotometer Turbidity

Viable Plate Count : 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 : Viable Plate Count Plate Counts: Perform serial dilutions of a sample

Viable Plate Count : Viable Plate Count Inoculate Petri plates from serial dilutions

Viable Plate Count : Viable Plate Count After incubation, count colonies on plates that have 25-250 colonies.

Bacterial Population Growth Curve : 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 : Bacteria Population Growth Curve

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Phases of the 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 : Bacteria Population Growth Curve

Culturing Obligate Intracellular Pathogens in the Laboratory : Culturing Obligate Intracellular Pathogens in the Laboratory

Culturing Fungi 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 : 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 : 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, 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 : 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 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, 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

Slide 49 : Alternation of membrane permeability Damage to proteins Damage to nucleic acids Actions of Microbial Control Agents

Slide 50 : 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

Slide 51 : Moist heat- denatures proteins Autoclave: Large pressure cooker Steam under pressure 15 psi, 121.5C, 20 minutes

Slide 52 : 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

Slide 53 : Radiation- damages DNA Ionizing radiation (X rays, gamma rays, electron beams) Non-ionizing radiation (UV) Ultrasonic waves Microwaves kill by heat; not especially antimicrobial

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Slide 55 : Filtration

Slide 56 : Gaseous Atmosphere altering the atmosphere in which the microorganisms are located Ex. Gas gangerene

Slide 57 : 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 : 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 : 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? : 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

Slide 61 : 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

Slide 62 : Using Chemical Agents to Inhibit Microbial Growth Evaluating a disinfectant Disk-diffusion method

Chemical Food Preservatives : 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

Slide 64 : Microbial Characteristics and Microbial Control

FINISHED… : FINISHED… Quiz Next Meeting!