Microbial Growth and Control

This is based on New Zealand Qualification Authority Unit 8034 entitled "Culture microorganisms and control microbial growth."

To view a power point presentation on this unit click on this link [1]

1.1

The description outlines the nutrient requirements of bacteria.

Bacteria as with all other living things have specific nutrient requirements. The main nutritional requirements of bacteria are water, a source of carbon for cell growth such as glucose, amino acids, and salts such as magnesium, phosphorus, nitrogen, and sulfur depending on the specific bacteria. Most of the bacterias are Chemoheterotrophs and uses organic compounds as the source of energy.Some of the bacteria makes a mutually beneficial interaction with other living organism to serve for the purpose of thier nutritional requirements for example: Bacteria "Rhizobia" and Leguminious Plants, this bacteria lives within the roots of plants such as soybeans, clover here they convert the atmospheric nitrogen to ammonia, which is used by plants as a nitrogen source. As the plants grow the product of their photosynthesis provide essential carbon sources for "Rhizobia". Bacteria also use the elemental traces such as Zn, Cu, Co, Mo. These are in the form of water, inorganic ions, small molecules, macromolecules.they take up these substance by membrane transport process.

The nutritional requirements of carbon,oxygen and hydrogen are satisfied together in almost all bacteria,and they are being classified according to the uptake of these macroelements.The microorganisms which can fix CO₂ ie;reduce it and incorporate in to organic molecules are called as Autotrophs.Heterotrophs are organisms that use reduced, performed organic molecules as carbon sources. A Prototroph is the organism which uses the same nutrients as all other organisms in its species uses.When a Prototroph lacks the ability to synthesize an essential nutrient due to mutation,and then it requires the nutrient or the precursor from the surroundings is called as Auxotroph. Organisms which use light energy as their energy source are called Phototrophs,and Chemotrophs obtain energy from the oxidation of chemical compounds. Organotrophs are organisms extracting hydrogen from the organic compounds.Lithotrophs use reduced inorganic substance from the rocks.

Growth Factors

They are required in small amounts by bacteria because they fulfill specific role in biosynthesis. Growth factors are organized into three categories.

1. Purines and pyrimidines: They are required for synthesis of nucleic acids(DNA and RNA).

2. Amino Acids: required for synthesis of proteins.

3. Vitamins: needed as coenzymes and functional groups of certain enzymes.

1.2

The description outlines the types of media used to culture bacteria.

There are two main groups of bacterial growth medium; these are broths and agar gels.Culture media provides not only a nutritional framework, but a support media on/in which they can survive (whether this be solid or liquid phase).[2] Agar gels can be designed to meet the essential nutritional requirements of a wide range of bacteria, or designed to limit some bacteria while not limiting or even stimulating the growth of other targeted bacteria.

Defined media or synthetic media has all of the macro and microelements and minerals and all of its components are known.Where in a Complex media the ingradients are of unknown chemical composition.Complex media contain undefined components like peptones,meat extarct,yeast extract.Peptones are proitien hydrolysates prepared by partial proteolytic digesion of meat,casein,gelatin,soyameal etc and it serves the carbon,energy and nitrogen.Beef extract contains aminoacids,peptides,nucleiotides and minerals;whereas the Yeast extract serves B vitamins and with nitrogen.Most used complex media are 1-nutrient broth,2-tryptic soy broth,3-MacConkey agar in which agar is the sulfated polymer composed mainly of D-galactose and 3,6-anhydro-L-galactose and D-glucuronic acid.Agar is well suited solidifying agent for complex media and it is usually extratced from a red algae.

Nutrient media is a non selective medium that contains all the necessary ingredients required, to enable the growth of a very broad range of different bacteria.

Selective, nutritionally defined or minimal media contain ingredients such as antibiotics, molecules or elements which will affected bacterial growth and multiplication depending on which environment specific bacteria thrive in, are stunted by or are killed by.

Enrichment Media When species of special interst is present in a small numbers ,then "Enrichment Media"is used, this media favours the growth of that species but not growth of others present in the mixed population.Enrichment techniques provide an environment,both chemical and physical ,results in increase number of scarce species.No inhibitory agent is used to prevent the growth of unwanted microorganism .

Enriched Media Contains extra nutrient for the growth of fastidious bacteria.

Diffrential / Indicator Media Divide the bacteria in to two or more groups based on the properties . e.g. Gram +ve and Gram -ve bacteria.

Transport Media In transport media pathogenic bacteria dies and pathogenic bacteria are overgrown by non-pathogenic. e.g. transport media for streptoccocus pyogenes - Pikes Media.

Bacteria that thrive in these heavily defined media are often defined as "fastidious organisms". Fastidious bacteria are often bacteria that fit the following catagories:
1. Recently Identified: Don't fall into a specific group so specific nutritional requirements are unknown.
2. Extremophilic: Bacteria that have evolved in extreme environments and require a very similar environment to propagate.

Microbiological assay media

Some of microorganisms can be used for the concentration of substances like antibiotics, minerals, vitamins etc. Many of antibiotics are resistant to blood serum or tissue fluids. Than in this type of assay is involves the measurement of growth inhibition caused by the antibiotics within the given time level of inhibition and it is proportional to the amount of drugs. Eg persons exposed to penicillin for long time unknowingly and treated with same antibiotics, and the patient becomes resistant to the penicillin.

Image taken by Radheyshyam N Bhagat UCOL National Diploma in Science 2008.(Antibiotic assay 27nov08)

2.1

Use of a closed system to grow micro organisms.

Micro organisms can be grown in closed systems for the purpose of study, which can include counting to assess it’s prevalence in a given sample, and to isolate individual target organisms for further study.

They are incubated in a closed culture vessel (Petri dish) with a batch of medium. Because no fresh medium is provided during incubation,nutrient concentration decline and concentration of waste increase.

The most common closed system method used for culturing organisms is the Petri dish, where nutrient rich agar is placed inside the Petri dish and then inoculated with a known or unknown micro organism, which is then incubated.


SPC agar plate (photograghed by D Beasley,NZ).

A 1ml water sample pre-treatment, collected from the town catchment area was added to this Petri dish which contained SPC agar. The pour plate method was used.
Using a closed system enabled us to contain the sample and provide the ideal environment for these organisms to grow.

Micro organisms are also commonly cultured in a closed system, where a nutrient rich liquid media is used.
In both methods water is always present, and is essential for microbial growth. Aseptic technique is used to avoid unwanted contamination from the working environment, and the organisms are contained within the closed system.

Essentially the micro organisms growing environment can be controlled. Samples can be incub of ated at various temperatures, different agars can be used, and pH can be altered. All of these factors facilitate the manipulation of micro organisms through controlled growth to further enable the isolation of a single species to be identified and studied.

The growth of microorganisms can be defined as the increase in cellular constituents.Growth results in an increse in cell number when microbes reproduce by budding or binary fission.But in Coenocytes ;organisms that are multinucletaed in which nuclear divisons are not accompanied by the cellular divisions-the growth results in increase in cellular size.The four phases in the life cycle of a microbe or microbial culture is called as growth curve.When microorganisms are introduced to afresh culture ,no spontaneous increase in cellnumber or mass occurs and this phase is called as Lag phase.It is in the Exponential or Log phase the microorganisms grow and divide at a maximal rate possible given by their geneticl potential,the nature of the medium and the conditions.in this phase microbes divide and multiply at regular intervals say expoentially,hence the name.When the microbial population reaches about 10^9 cells per ml,the growth ceases and the growth curve becomes horizontal.This is called the Staionary phase and it occurs due to the insufficieny in nutrients.Death phase is the final stage in growth in which the microorganisms deprived of essential nutrients die due to the build up of toxic waste products and lack of nutrients.

2.2

The description outlines the culture of microorganisms under appropriate condition in accordance with laboratory protocol.

Pipette should be filled with use of pipette aids and operated in such away to aviod creating aerosols. Hypodermics syringe and needle should be used when ever necessary and then with care. Aqueous aerosol is a gaseous suspension of liquid or solid particles should be away from flames and, removal of closures from shaken culture tubes and contaminated loop should be away from contaminating materials. Mixing of the samples should be appropriate and colonies should be cultured for a specific time period and at specific temprature

3.1

The description outlines the use of physical procedures to limit microbial growth.

The mainly employed physical agents are heat,filtration,ultraviolet radiation, ionizing radiation , low temprature and desiccation.

HEAT

The shortest time required to kill all the organisms in a suspension at a specific temperatures at defined conditions are called as TDT or thermal death time.But nowadays DRT or Decimal reduction time or D value is used which is the time required to kill the 90% of the microbial population or spores in a sample at given specific temperature.Moist heat sterilization usually carried out above 100 degree C in order to destroy bacterial endospores. An Autoclave is a device used to employ the steam sterilization.Pasteurization is a technique used for sterilizing diary products using controlled heating well below the boiling temperature.Many objects are sterilized using dry heat sterilization with the absence of water.

FILTRATION

The simplest way of removing microbes from solutions not by destroying them is Filtarion.Depth filters are the older versions which contains fibrous or membranous pores bonded to form a thick layer filled with channels of small diameter.The solution containing microorganisms is sucked through this layer under vacuum.Membarne filters have substituted the Depth filters as they can be produced into a wide variety of pore sizeup to 0.2um in diameter.The microbial cells are removed by physical screening or Entrapment or Microencapsulation.Laminar flow biological saftey cabinets are using high efficiency particulate air or HEPA which can remove almost 100% of the 0.3um size particles.

RADIATION

The Ultraviolet radiation of wavelength 260 nm are used in very specific cases because it cannot penetrate glass,dirt films and water and it is harmful to humans as the continous exposure causes genetic mutaion or DNA.

The Ionizing radiation like the Gamma rays from a Cobalt 60 source is used in the cold sterilization of abtibiotics and hormones because Cobalt 60 have high penetration for solids and liquid and destroy microorganisns by the formation of hydroxyl free radicals and peroxides from water and other substances.It is used in destroying pathogens in poultry an extending the shelf life of seafoods and fruits e.g.strawberries,apples.And it is strongly recommended by the US Food and Drug Administration also.

LOW TEMPRATURE

Deep freezing is the method used to slow the growth of microorganisms. This method is bacteriostatic and commonly used to preserve the drugs and food.

DESICCATION

1. Lyophilization: method used to arrest the growth of microorganisms and used for long term preservation of microbes.

2. Osmotic Pressure: method for plasmolysis of microbes ( loss of water) . This method is used for food preservation.

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3.2

The description outlines the use of chemical agents to limit microbial growth.

Antiseptics are the chemical agents apply to tissue or fluids to prevent the infection by killing or inhibiting pathogenic growth.These are temporary prevention and not so toxic.For example; (1)Alcohol:-Ethyl alcohols having the concentrations of 70 and 90% are effective against the vegetative forms of microorganisms .The bactericidal property of alcohols increases as the length of carbon chain increases.Alcohols are protien denaturants which accounts to a large extent for their antimicrobial activity.Alcohols are also lipid solvents,thus damaging the lipid structures inside the microbial cell membrane.

(2)Phenols:-These are the carbolic acid having distinction of being one of the first chemical agent used as an antiseptic agent.Phenol and phenolic compounds damage microbial cells by altering the normal selective permiability of cytoplasmic membrane,causing leakage of essential intracellular substances.These chemical also denature and inactivate proteins such as enzymes.

Germicides chemical agents that kill or inhibit growth of microorganisms and are sufficiently nontoxic to be applied to living tissues.

Sanitizers reduce , but may not eliminate, microbial numbers to a " safe " level- water soluble.

Disinfactants are agents used to carryout disinfaction.They kill, inhibit or remove the microorganisms that cause disease,but not kill thier spores and mostly used on inanimate objects.These chemicals also kill or inhibit the growth of non pathogenic organisms as well.

Sterilant Sterlization is the process of destroying or removing all forms of microbial from an object or a specimen.Thus a sterile item is one free of all microorganisms and Sterilant is a chemical agent that accomplishes sterlization.e.g.Ethylene oxide,Formaldehyde.

4.1

The description outlines the effect of selected factors on the efficacy of antimicrobial agents.

There are two main methods of controlling microbial growth; this is with the use of chemicals such as disinfectants or through physical action such as temperature (high or low). Microbes, like all living things, all have essential environmental conditions which are needed to properly function. Controlling microbes by physical means, usually involves removing one or more of these essentially required living conditions, such as water, correct temperature or pH, or by increasing radiation(ionizing and Non-Ionizing) or UV,Filtration like High Efficiancy Particulate Air(HEPA)filters,population size,intesity or concentration of microbicidal agent,population composition,time of exposure to the microbial agent,nature of material.

(1)Population Size: The more will be the size of population ,more it will take to kill ,than smaller population.

(2)Intensity or concentration of the microbicidal agent: The lower the intesity or concentration ,the longer it takes to kill a microbial population.

(3)Population Composition:The effectiveness of an agent varies greatly with the nature of organism being treated because microorganism differ markedly susceptibility. e.g Mycobacterium tuberculosis is much more resistant to antimicrobial agent than most other bacteria.

(4)Time of exposure to microbial agent: the longer time a population is exposed to a microbial agent,the more are killed.

(5)Nature of material:different characterstics of the material may affect the rate of killing of cells caused by microbicidal agent.

(6) Temperature at which microorganisms are exposed to microbial agent: At higher temperature one can kill large amount of bacteria.

(7) Characteristic of microorganisms : Gram positive bacteria are more resistant to heat than gram negative and some time gram negative bacteria are more resistant to heat than gram positive.

(8) pH : For some antimicrobial agents decrease in pH make them more effective while other agents become inactive as the pH drops.

Different micro organisms require different environmental conditions. Once the micro organism is identified, the best method of control can be chosen. An example of this would be cleaning a food preparation surface with bleach to eliminate a wide range of bacteria. A chemical method such as this will kill or hugely limit most types of microbial growth. The main groups of chemical control agents are; antibiotics, antiseptics, disinfectants and preservatives.

Antibiotics are chemicals which prevent the growth of harmful micro organisms, and are usually ingested or administered intravenously. Antibiotics target the pathogenic organism, leaving the host cell unharmed.

Antiseptics are manufactured substances which kill or severely inhibit the growth of microbes. These are for topical use only and can be harmful in high concentrations or if taken orally.

Disinfectants are chemicals which are used to severely reduce or eliminate microbes from surfaces. Some disinfectants such as hydrogen peroxide can also be used as antiseptics when in low concentrations of 3% and less.

Preservatives are used to limit microbial growth in many areas, such as timber, food and also to preserve corpses before they are buried. Chemicals such as salt, nitrate and sulphates can be used as preservatives.

5.1-5.2

The description outlines the mode of action of antibiotics & The description notes the factors which limit an antibiotic’s effectiveness (respectively).

There are many different types of antibiotics, with various modes of action, all of which disrupt the normal function of effected micro organisms (bacteria, fungus or protozoa).

Amoxicillin is one example of a widely used antibiotic which is effective against a range of gram positives such as Streptococcus, and Staphylococcus, and a limited number of gram negatives such as Salmonella. It is in the group called lactams. Amoxicillin disrupts the normal synthesis of the cell wall, preventing the cross linking of the peptidoglycan protective shell, which is relatively thick in gram positives. This then causes the growth of the peptidoglycan layer to cease, while the bacterial cell continues to grow until it bursts.

Tetracyclines are the antibiotics which inhibit the bacterial growth or production by binding to the small(30s)ribosomal subunits and interfering with the attachment amino acyl-transfer RNA to ribosome.Unlike the aminoglycosides,tetracyclines binds to both eucaryotic and procaryotic ribosome,but they are more selective to procaryotes because eucaryotic membrane are less permiable to antibiotics.They are mainly produced by species of the genus streptomyces.They are active against gram-positive and gram-negative bacteria.

Some bacteria, such as Staphylococcus, mentioned above have the ability to develop resistance against penicillin derived antibiotics such as amoxicillin, by producing an enzyme called penicillinase which disables the antibiotic. Penicillins do not work against fungi or viruses. Another antibiotic, Doxycycline, one of many in the tetracycline group, prevents tRNA and ribosome's from functioning together and so prevents cell growth. Bacteria have developed resistance to antibiotics in this tetracycline group, in several ways. One adaptation bacteria have made to counteract this class of antibiotic is to produce protective proteins, that protect the bacterial ribosome's.

Follow this link to see the various modes of action, of different antibiotic groups. [4]

Mode of action of antibiotics on Cytoplasmic Membrane

Antibiotic such as Polymyxins have ability to damage the cytoplasmic membrane of bacteria. Polymyxins are bactericidal because they disrupt the phospholipids that make up the structure of the membrane. This destroys normal permeability characteristics of cell membrane. So that essential substances leak from cells, resulting in cell death.

'Antibiotics and Mode of action'

Antibiotics	Mode of action	Active against
Penicillins, Cephalosporins, Monobactams (Beta-Lactams)	Inhibit cell wall synthesis http://www.youtube.com/watch?v=gTWiaH_oCCY	Gram positive bacteria,Gram negative bacteria causing respiratory, intestinal and urinary infections
Streptomycin, Spectinimycin, Neomycin, Kanamycin, Gentamicin (Aminoglycosides)	Induce abnormal protein synthesis	Tuberculosis infection Penicillin resistant Neisseria gonorrhoeae, Inhibits intestinal bacteria,Gram negative bacteria and Gram positive
Tetracyclines	Interfere with protein synthesis	Broad spectrum bacteria
Erythromycin, Lincomycin (Macrolides)	Interferes with protein synthesis	Gram positive bacteria
Chloramphenicol	Interferes with protein synthesis	Broad spectrum bacteria
Vancomycin	Interferes with protein synthesis	Gram positive including penicillinase producing staphylococci and enterococci
Viomycin	Interferes with protein synthesis	Tuberculosis infection
Rifamycin	Interferes with protein synthesis	Tuberculosis infection
Polyenes Nystatin (Antifungal)	Damage Cell membrane	Fungal infections, particularly oral, skin, intestinal and vaginal lesions http://video.google.com/videoplay?docid=7791741076863572309
Amphotericin B (Antifungal)	Interferes with membrane function	Deep seated mycotic infection
Griseofulvin (Antifungal)	Damage Cell membrane	Fungal infections
Sulfonamides , Sulfones , Trimethoprim , Pyrimethamine , Ethambutol	Interfere with intermediary metabolism	Urinary infections and nocardia infections.
Fluoroquinolones-ciprofloxacin	Interfere with DNA synthesis	Urinary tract infections , Gonorrhoea , Chancroid

Tuberculosis treatment and incompliance

http://www.youtube.com/watch?v=Y7VkEXuNaiU

6

Effectiveness of an antibiotic

Antibiotics are the molecules produced by microbes that inhibit the growth or kill, the other microbes. They are normally used to treat the bacterial infection those that are selectively toxic to bacterial cells and do not harm the host cells. They target the sites such as Peptidoglycan, ribosomes, nucleic acid synthesis which are different for prokaryotic and eukaryotic cells. The effectiveness of antibiotic is reduced by development of bacterial antibiotic resistant. This resistance occured due the change in structure of bacteria because of long time usage of specific antibiotic.

6.1

The description outlines the selection of appropriate organism in accordance with laboratory prtocol.

The antibiotics are the therapeutic prepartions which dissects metabolic processes by inhibiting or blocking the specific steps in microrganisms or the host cell.To be a successful antibiotic it must kill or inhibit the microbial pathogen while damaging the host cell as little as posssible.The degree of selective toxicity can be expressed in terms of the Therapeutic dose that is the drug level required for the clinical treatment of a particular infection.And the toxic dose that is the drug level at which the agent becomes too toxic for the host cell.Many of the antibiotic agents can cause undesired effects in the host cell called as Side effects.Drugs vary in their effectiveness as Narrow Spectrum Drugs which are effective only against a limited variety of pathogens and Broad Spectrum Drugs which attack many different kinds of pathogens.And antibiotics can be classified as antibacterial,antifungal,antiprotozoanand antiviarl depending on the general microbial group they act against.But some agents like Sulfinamides are active against bacteria and protozoa.In pratice the antibiotic is adminstered and changes in cell function are monitored.The results must be interpreted also.Because not all microorganisms respond to a same way to a particular drug.

6.2

The disc difffusion test is carried out in accordance with lab protocol .

Disc diffusion test is carried out to to determine the effectiveness of a antimicrobial agent against a specific rapidly growing aerobic or faculative pathogen like Staphylococccus or Pseudomonas. If there is no growth of bacteria it means bacteria is sensitive to antibiotic and their growth indicates the resistance.

References

Re: Why is it so hard to culture most bacteria. (2001). madsci.org. Retrieved September 30, 2008. Source: madsci.org forums [http://www.madsci.org/posts/archives/2001-03/984870351.Mi.r.html ]

Bacteria. (2008). In Encyclopædia Britannica. Retrieved September 22, 2008, from Encyclopædia Britannica Online: [http://www.britannica.com/EBchecked/topic/48203/bacteria ]

Endospores and Endospore Staining. Retrieved September 5,2008, source page: [http://howie.myweb.uga.edu/structure.html ]

A-level Applied Science/The Role of the Pathology Service/Microbiology. Retrieved October 16, 2008, from Wikibooks, the open-content textbooks collection: [http://en.wikibooks.org/wiki/Alevel_Applied_Science/The_Role_of_the_Pathology_Service/Microbiology ]
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Microbiology by J.Nicklein, K.graeme-Cook, T.Paget &R. Killington

Microbiology by LANSING M PRESCOTT,JOHN P HARLEY,DONALD A KLEIN