Environmental Microbiology -Laboratory Manual- prepared for Environmental Microbiology

Environmental Microbiology

-Laboratory

Manual-prepared for Environmental Microbiology

III

Microorganism Growth Factors Controlling Growth

anindrya.pahlawan@ymail.com

E N V I R O N M E N T A L M A N A G E M E N T T E C H N O L O G Y

MICROBIAL GROWTH

GROWTH

• In biophysics cells are open systems far from a thermodynamic equilibrium, exchange material and

energy with their environment, and especially exhibit a large outfow of entropy.

• In chemical engineering  growth is referred to as an increasing amount of biocatalyst

• Mathematical  growth are restricted to a couple of equations employing hyperbolic and exponential terms.

• Growth is usually considered as an increase of cell material expressed in terms of mass or cell number.

Microbial Nutrition

• Why is nutrition important?

▫

The hundreds of chemical compounds

present inside a living cell are formed

from nutrients.

This slide is taken from : MICR 300 : Microbiology, California State of University Macronutrients : elements required in fairly

large amounts

Micronutrients : metals and organic

Main Macronutrients

• _{Carbon (C, 50% of dry weight) and nitrogen}

(N, 12% of dry weight)

• _{Autotrophs are able to build all of their cellular}

organic molecules from carbon dioxide

• _{Nitrogen mainly incorporated in proteins,}

nucleic acids

• Most Bacteria can use NH₃ and many can also use NO₃

-• _{Nitrogen fxers can utilize atmospheric nitrogen}

(N₂)

Other Macronutrients

• _{Phosphate (P), sulfur (S), potassium (K),}

magnesium (Mg), calcium (Ca), sodium (Na), iron (Fe)

• _{Iron plays a major role in cellular respiration,}

being a key component of cytochromes and iron-sulfur proteins involved in electron

transport.

• _{Siderophores : Iron-binding agents that}

cells produce to obtain iron from various insoluble minerals.

Micronutrients

Need very little amount but critical to cell

function.

Often used as enzyme cofactors

Growth factors

Culture Media: Composition

• _{Culture media supply the nutritional}

needs of microorganisms

▫ _{defned medium : precise amounts of highly}

purifed chemicals

▫ _{complex medium(or undefned) : highly}

nutritious substances.

• _{Inclinical microbilogy,}

▫ _{Selective : contains compunds that selectively}

inhibit

▫ _{Diferential: contains indicator}

▫ _{terms that describe media used for the isolation}

of particular species or for comparative studies of microorganisms.

Culture Media: Physical Properties

• Liquid

▫ Bouillon or broth

• Solid

▫ _{Addition of a gelling agent (typically 1% agar) to}

liquid media

▫ Immobilize cells, allowing them to grow and form visible, isolated masses called colonies (Figure 5.2).

• Semisolid

▫ Reduced amount of agar added

▫ Allows motile microorganism to spread

Bacterial Colonies on Solid Media

S. marcescens (Mac)

P. aeruginosa (TSA)

S. flexneri (Mac)

Laboratory Culture of

Microorganisms

• _{Microorganisms can be grown in the}

laboratory in culture media containing the nutrients they require.

• _{Successful cultivation and maintenance}

of pure cultures of microorganisms can be done only if aseptic technique is

practiced to prevent contamination by other microorganisms.

• _{Microbial growth}

involves an increase in the number of

cells.

• _{Growth of most}

microorganisms occurs by the

process of binary fssion

Cell Growth and Binary Fission

Microbial Growth

Peptidoglycan layer

• _Microbial

populations show a characteristic type of growth pattern called exponential growth, which is best seen by

plotting the number of cells over time on a semi- logarithmic graph.

Microbial Growth Pattern

Growth Curve

• _{Microorganisms show a characteristic growth}

pattern (Figure 6.8) when inoculated into a fresh culture medium.

Measuring Microbial Growth

• _{Growth is measured by the change in the}

number of cells over time.

▫ _{Cell counts done microscopically measure the}

total number of cells in a population

▫ _{whereas viable cell counts (plate counts)}

measure only the living, reproducing population.

Environmental Efects on Bacterial

Growth

• _Temperature

• _pH

• _{Osmotic pressure} • _{Oxygen classes}

Temperature and Microbial Growth

• _{Cardinal temperatures}

▫ _minimum ▫ optimum

▫ _maximum

• Temperature is a major environmental factor controlling microbial growth.

Classifcation of Microorganisms by Temperature Requirements

pH and Microbial Growth

• _{The acidity or alkalinity of an environment can}

greatly afect microbial growth.

• _{Most organisms grow best between pH 6 and 8, but}

some organisms have evolved to grow best at low or high pH. The internal pH of a cell must stay

relatively close to neutral even though the external pH is highly acidic or basic.

▫ _{Acidophiles : organisms that grow best at low pH} ▫ _{Alkaliphiles : organismsa that grow best at high pH}

Osmotic Efects on Microbial Growth

• _{Osmotic pressure depends on the surrounding}

solute concentration and water availability

• _{Water availability is generally expressed in}

physical terms such as water activity

• _{Water activity is the ratio of the vapor}

pressure of the air in equilibrium with a

substance or solution to the vapor pressure of pure water.

Halophiles and Related Organisms

• _{In nature, osmotic efects are of interest mainly in}

habitats with high salt environments that have reduced water availability

• _{Halophiles : have evolved to grow best at}

reduced water potential, and some (extreme halophiles) even require high levels of salts for growth.

• _{Halotolerant : can tolerate some reduction in the}

water activity of their environment but generally grow best in the absence of the added solute

• _{Xerophiles : are able to grow in very dry}

environments

Oxygen and Microbial Growth

• _{Aerobes :}

▫ _{Obligate : require oxygen to grow}

▫ _{Facultative : can live with or without oxygen but}

grow better with oxygen

▫ _{Microaerphiles : require reduced level of oxygen}

• _{Anaerobes :}

▫ _{Aerotolerant anaerobes : can tolerate oxygen}

but grow better without oxygen.

▫ _{Obligate : do not require oxygen. Obligate}

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Heat

Heat: Is the most practical, eficient, and

inexpensive method of sterilization of those inanimate objects and materials that can withstand high temperatures.

Two factors, temperature and time, determine the efectiveness of heat for sterilization.

Sterilization is the process whereby all viable microbes including spores are removed or killed.

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Heat (2)

a. Dry: At 160 to 165° C for 2 hours or at 170 to 180°C for 1 hour.

- Red heat and incineration (burning): Direct exposure of material to fame till it becomes red. E.g., culture loops.

- Direct faming: Passing material over fame many times without reaching redness. E.g., faming mouths of bottles, slides, fasks and culture tubes.

- Hot air oven: Supplied with a fan from inside to distribute hot air in all chamber. It has temperature thermostat. It is used widely in

hospitals, clinics, and laboratories. E.g., test tubes, glass pipettes, scissors, blades.

b. Moist: Heat applied in the presence of moisture, as in boiling or steaming, is safer and more efective than dry heat, and can be accomplished at a lower temperature for 30 min.; thus, it is less

destructive to many materials. Moist heat causes proteins to coagulate (as occurs when eggs are hard boiled)

- Boiling and steaming: At 100° C. clean articles made of metal and glass, such as syringes, needles may be disinfected by boiling for 30 minutes. Boiling is not always efective because heat- resistance bacterial

endospores, mycobacteria and viruses may be present.

- Autoclaving (steam under pressure):

An autoclave is like a large metal pressure cooker that uses steam under pressure to completely destroy all microbial life.

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Cold

2. Cold: Most microbes are not killed by cold temperatures and freezing, but their

metabolic activities are slowed, greatly inhibiting their growth.

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Radiation

Radiation:

a. Nonionizing radiation:

- ultraviolet (UV) rays (has low degree of penetration): A

ultraviolet lamp (germicidal lamp) is useful for reducing the number of microorganisms in the air and on

surfaces.

They do, however, penetrate cell and, thus, can cause damage to DNA. When this occurs, genes may be so severely damaged that the cell dies. Many biologic

materials, such as toxins, and vaccines, are sterilized with UV rays.

b. Ionizing radiation:

- X-rays and gamma rays (has high degree of

penetration): Are used in industry for sterilization of plastic catheters, syringes, surgical equipments,

preparation of vaccines…etc.

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Filtration

4. Filtration: Is the passage of a liquid or gas through a flter with pores small enough to allow microbes to pass. This method used for sterilization of serum, hormones, antibiotic solution.

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Gases

5. Gases (ethylene oxide, propylene oxide…etc): Suitable for plastics, hormones, surgical

dressing, all antibiotics and thermo labile powder.

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Antiseptics

1. Antiseptics: chemicals inhibit the growth or kill microbes on living tissues like human skin and mucus membranes.

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Disinfectants

1. Disinfectants: chemicals inhibit the growth or kill microbes.

- Factors that determine the efectiveness of any disinfectant:

a. Time.

b. Temperature. c. Concentration.

d. Type and number of microbes. e. Presence of spores.

f. Presence of proteins in feces, blood, vomitus, pus.

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Disinfectants (2)

Characteristics of good disinfectant:

Rapid action, easy to use.

Wide range of action.

_{Good penetration.}

Capability of mixing with water.

Activity in organic matter (like blood, feces, vomit)

_{Resistance to decomposition.} _{Nonstaining and noncorrosive.} _Odorless.

_{Stable in various temperature and light.} Cheep.

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Disinfectants (3)

- Kinds of Disinfectants:

a. Alcohol: 70% to disinfect skin and thermometer. b. Phenolics:

d. Iodine (povidone):

for skin disinfection (available as a tincture ,2% iodine with 70% alcohol).

e. H2O2 (hydrogene peroxide)

3 – 6 % for wounds, ulcers, and mouth wash.

f. Formaldehyde: for rubber, leather, shoes, books, and blankets.

CHEMOTHEURAPEUTICS

USING ANTI MICROBIAL AGENTS TO CONTROL MICROBIAL GROWTH IN VIVO

Nabeel Al-Mawajdeh RN.MCS

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Using Chemotherapeutic agents

- Chemotherapeutic agent is any chemical

(drug) used to treat an infectious disease, either by inhibiting or killing pathogens in vivo.

a. Antifungal agents are used to treat fungal diseases.

b. Antiprotozoal agents are used to treat protozoal diseases.

c. Antiviral agents are used to treat viral diseases. d. Antibiotics

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Using Chemotherapeutic agents

(Cont’d)

d. Antibiotics are substances produced by

microorganisms (usually a soil organism) that efective in killing or inhibiting the growth of other microorganisms. Some antibiotics (e.g., penicillin and cephalosporin) are produced by molds, whereas others (e.g., tetracycline,

erythromycin, and chloramphenicol) are produced by bacteria.

Many antibiotics have been chemically modifed

to kill a wider variety of pathogens or reduce

side efects; these modifed antibiotics are called semisynthetic antibiotics.

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Ideal Qualities Of Antimicrobial

Agents

1. Antimicrobial agents should have selective toxicity

for microorganisms. E.g., it can kill or inhibit the

growth of a microorganism in concentrations that

are not harmful to the cells of the host.

Therapeutic index= large amount of antimicrobial can be given without harm

Lowest dose that can kill microorganism

The higher the therapeutic index the better the antimicrobial.

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Ideal Qualities Of Antimicrobial Agents

(Cont’d)

2. Should Kill or inhibit the growth of pathogens.

3. Cause no damage to the host.

4. Be stable when stored in solid or liquid form.

5. Remain in specifc tissues in the body long

enough

to be efective.

6. Kill the pathogens before the mutate and

become resistant mutant pathogens.

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Mechanisms of Resistance to

Antimicrobial Agents

1. The organism produces enzymes that destroy

the drug. E.g., production of beta – lactamases that destroys penicillin.

2. The organism changes its permeability to the

drug, by modifcation of protein in the outer cell membranes, thus impairing its active

transport into the cell e.g., resistance to polymyxins.

3. The organism develops an altered receptor site

for the drug e.g., resistance to aminoglycosides is associated with alteration of a specifc

protein in the 30s subunit of the bacterial ribosome that serves as a binding site in susceptible organisms.

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Mechanisms of Resistance to Antimicrobial Agents (Cont’d)

4. The organism develops an altered metabolic

pathway that bypasses the reaction inhibited by the drug e.g., sulphonamide- resistant bacteria acquire the ability to use performed folic acid with no need for extracellular PABA (p-

aminobenzoic acid)

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Clinical Use of Antibiotics

1. Antibiotics should not be given for trivial infections.

2. Treatment should be based on a clear clinical

and bacteriological diagnosis. According to results of antibiotic sensitivity testing in vitro.

3. Antibiotics for systemic treatment should be

given in full therapeutic doses for adequate period.

4. Combined therapy with two or more antibiotics

is required in some conditions. E.g,

a. Serious resistant infections e.g., infective endocarditis or meningitis.

b. In treatment of tuberculosis 2 or 3 drugs are given by lowering the dose of each to decrease toxic efects of drugs. c. Severe mixed infections e.g., peritonitis following

perforation of the colon.

Exp.8 -Temperature

a. Efect of temperature on bacteria and yeast cells b. Efect of temperature on vegetative and spore cells

Durham Tube

• _{Durham tubes are used in}

microbiology to detect production of gas by microorganisms.

• This small tube is initially flled with the solution in which the microorganism is to be grown. If gas is produced after

inoculation and

8.B. Efect of temperature on

vegetative and spore cells of bacteria,

yeast, and fungi

m

Exp. 13 Disinfectant and

Antiseptic

a. Alcohol efectivity evaluation

b. Antiseptic evaluation using blotter

13.A. Alcohol efectivity

evaluation

_{• A = left thumb without}

alcohol

• _{B = left thumb without}

alcohol

• _{C = right thumb}

without alcohol

• _{D = right thumb with}

alcohol

• _{Incubate the dish}

A B

13.C. Efectivity of hand washing using

soap

RATIONAL USE OF

MEDICINE



_Use