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4. Cultivation of bacteria Cultivation process Cultivation is the process of propagating organisms by providing proper environmental conditions. (main reason for this is to isolate a pure culture and identify a possible pathogenic bacteria from clinical or pathological samples) In order to grow, an...

4. Cultivation of bacteria Cultivation process Cultivation is the process of propagating organisms by providing proper environmental conditions. (main reason for this is to isolate a pure culture and identify a possible pathogenic bacteria from clinical or pathological samples) In order to grow, an organism requires all of the elements in its organic matter and the full complement of ions required for energetics and catalysis and there must be a source of energy (see previous lecture). Factors that must be controlled during growth include the nutrients, pH, temperature, aeration, salt concentration, and ionic strength. Cultivation in a laboratory setting is done by using a suitable medium. (medium is a specifically prepared environment in the laboratory which ensures the preservation, growth and reproduction of bacteria) Media must meet the following requirements: 1)Must containt all the necessary chemical components that bacteria need; 2)They must have a certain (usually neutral or weakly alkaline) environmental pH; 3)They must be sterile. Typically media consist of: Nutrients (proteins, amino acids) Energy source (carbohydrates) Essential metals and minerals Buffering agents (phosphate , acetate) (Indicators for pH change)(phenolred) (Selective agents) (antibiotics) Gelling compound – agar-agar. Bacterial culture media can be classified based on composition, consistency, and purpose. Bacterial culture media based on consistency Liquid media – contains nutrients, but don’t have a trace of gelling agents such as gelatin or agar. Examples – nutrient broth, tryptic soy broth, brain-heart infusion broth, selenite F broth etc. (glycose broth, alkaline peptone water) Broth medium serves various purposes: propagation of many organisms, accumulation of bacterial toxic end product, fermentation studies etc) Semi-solid media – prepared with agar at 0.5% or less concentration. Used for cultivation of microaerophilic bacteria or for determining bacterial motility. Examples – motility test medium, Stuart’s and Amies transport media etc. Solid media – contain agar at a concentration of 1.5-2.0% or some other primarily inert solidifying agent. On solid media bacteria grow in a physically informative ways by forming visible colonies (this aids in identification process). Examples – nutrient agar, blood agar, MacConkey agar, Baird-Parker agar etc. Agar-agar Agar-agar – a polysaccharide extracted from the cell walls of red algae. Agar-agar is inert and non-nutritious, therefore the agar must be impregnated with nutrients such as beef extract and peptone in order to support life. Agar melts at around 97-100 °C and solidifies at 42 °C. If the medium contains 1-5% of agar-agar, the name of the medium also has «agar» in it. Test tube slanting, Bacterial culture media based on composition Chemically defined media – exact amounts of highly purified inorganic and organic ingredients are known. Example – citrate broth. (mainly for experimental purposes or diagnostic bacteriology) Complex media – prepared using digests of microbial, animal or plant products (casein, beef extract, yeast extract etc.). These are highly nutritious yet impure substances; thus, the exact composition of the medium is not known. Example – tryptic soy broth. General-purpose (basal) media – capable of sustaining growth of large variety, less fastidious bacteria. Example nutrient agar, generally used for primary isolation for micoorganisms Bacterial culture based on functional use Enriched media – basal media with additional nutrients (such as blood, serum, egg yolk etc.). Supports growth of most bacteria including many fastidious species. Examples – blood agar, chocolate agar, Loeffler’s medium etc. Blood agar 5-10% blood to general purpose base. Important in the clinical medium many bacteria can haemolyse blood partially or completely. Haemolyses patterns can help differentiate and identify certain bacterial pathogens. Chocolate agar heating blood agar which ruptures the red blood cells and releases nutrients that aid in the growth of fastidious bacteria such as neseria? and hemophilia species. The ruptured red blood cells give the plate a chocolate brown colour. Not used for haemolyses. Differential (indicator) media – designed to give a presumptive identification of bacterial colonies due to the biochemical reactions in the media. Often contain a fermentable sugars plus a pH indicator that gives a color change in the media. Useful in diagnostic bacteriology. Examples – Bromocresol purple lactose agar used for enterobacterio?,nonselective Cysteine Lactose Electrolyte deficient (CLED) agar urinary pathogens, nonselective Selective media – contains inhibitory substances that prevent the growth of unwanted bacterial species and promotes the growth of a particular bacterium or group of bacteria. Used in diagnostic bacteriology. Examples – Lowenstein Jensen medium for mycobacteria, 6.5% NaCl broth for enterococci. Enrichment media –not enriched media!!! liquid media that permit the growth and detection of a particular bacterium, which may have made up only a small proportion of the bacteria in the original inoculum. Selective, inhibits the growth of normal flora permits the growth of small bacterium until it reaches the wanted numbers and later they can be subcultured to selective differential media Example – selenite broth for the selection of salmonellae, Rappaport-Vassiliadis soya peptone broth for salmonellae, Fraser broth for Listeria. Selective-differential media – contains both selective and differential agents (promotes the growth of a specific type of bacteria and helps to differentiate between genera and/or species). Examples – Mannitol salt agar inhibits the growth of other bacteria execpt staphylococci, MacConkey agar contains crystal violet which inhibits gram postive bacteria contains lactose (helps to differentiate between fermenting and non fermenting bacteria), Baird-Parker agar, XLD agar etc. Transport media – ensures the viability of potential pathogens in the sample and prevents drying (desiccation) of a specimen and overgrowth of contaminating organisms or commensals while the sample is being transported to the laboratory. Examples – Cary Blair transport medium, Stuart’s transport medium (semisolid), Amies transport medium with/without charcoal (semisolid, addition of charocoal serves to neutralise the materials that are toxic to sensitive pathogens) Other special media for anaerobes, need less oxygenation concentration and extra nutrients (Thioglycollate broth, Robertson’s cooked meat medium) or assay media Miller Hilton agar (for antibiogram, bacterial enumeration etc.) Types of haemolysis Alpha haemolysis – under the colonies, greenish color. Caused by H2O2, hemoglobin is oxidized to methemoglobin. Incomplete and partial. Beta haemolysis – complete lysis of RBCs under and around the colonies due to production of exotoxins – streptolysins. The area appears lightened (yellow) and clear. Gamma haemolysis – the organism does not induce haemolysis and the agar under and around the colonies is unchanged. Nonhaemolytic. Preservation of bacteria Subculturing is a short-term preservation of bacteria. Limitations to this procedure include death of cells, risk of contamination and mutations. Long-term methods include paraffin method, freeze-drying (lyophilization), freezing at -70 °C and ultra-freezing (cryopreservation) in liquid nitrogen at -196 °C. If correctly used can be used for 30 years Unculturable bacteria Unculturable bacteria are metabolically active in their native environment, but are unable to proliferate in laboratory media. “Unculturable” doesn’t mean there’s no bacteria in the sample. For example, intracellular bacteria (like Rickettsia, Coxiella, Chlamydia, Ehrlichia etc.) cannot be grown in liquid or solid media, but can be cultivated in cell cultures. 5. Physical methods of microbial control General concepts Many infectious agents can be easily transmitted on inanimate objects, so steriliztion and antiseptis are important Sterilization – all viable microorganisms are eliminated or destroyed (including viruses and endospores). No levels in sterility. Disinfection – destruction or significant reduction of microorganisms (excluding endospores) on nonliving materials by physical process or chemical agent. (effective against vegetative cells) Antisepsis – inactivation and destruction of vegetative microbial forms on animals or living tissues by chemical means (antiseptics=disinfectants). Asepsis – absence of bacteria, viruses and other microorganisms. Related to the practice of preventing the introduction of harmful microorganisms into the body (for example, wearing personal protective equipment etc.). Bactericidal – a chemical that destroys vegetative bacterial cells. Virucidal – a chemical that destroys or inactivates viruses. Fungicidal – a chemical that destroys fungi. Sporicidal – a chemical that destroys bacterial endospores. Germicidal/microbicidal – a chemical that kills microorganisms. Bacteriostatic – inhibition of multiplication or growth of bacteria. Fungistatic – inhibition of fungal growth. Effects of control methods on bacteria The adverse effects of physical or chemical agents on bacteria manifest in the following ways: All produce damaging effects to one or more cellular structures Cell-wall injury – lysis of the cell wall leaves the wall-less cell (called protoplast) unprotected and susceptible to osmotic damage, hypotonic environment will cause lysis of vulnerable protoplast. Certain agents inhibit cell wall synthesis which is essential during microbial cell reproduction and failure synthesize a missing segment will result an unprotected cell wall) Cell membrane damage – lysis of the membrane leads to immediate cell death. If only the selective nature of membrane is affected, there could be loss of essential cellular molecules or interference with the uptake of nutrients – metabolic processes will be adversely affected. Alteration of the colloidal state of cytoplasm – certain agents cause denaturing of cytoplasmic proteins → enzyme inactivation → cellular death. (irreveresbly rupturing these molecular bones of proteins and rendering them biologically inactive) Inactivation of cellular enzymes – enzymes can be inactivated competitively (reversible, occurs when a natural substrate is forced to compete for the active side of an enzyme surface with a chemically similar molecular substrate which can block the enzymes ability to create end products ) or noncompetitively (irreversible, occurs following the application of some physical agents). Interference with the structure and function of the DNA molecule – some agents have an affinity for DNA and cause breakage or distortion of the molecule → interference with protein synthesis and replication. Heat application Common way to destroy microorganisms Factors affecting sterilization with heat: Type of heat (both dry and moist but moist kills cells more rapidly and at lower temperature than dry heat) Temperature and time (higher the temperature the shorter the exposition time) Number of microorganisms (the more microbes the more it takes time) Type of microorganisms (some microbes are more heat resistant than others Type of material (some materials cannot be exposed to high temperatures) Presence of organic material (increases exposition time) Microbes exhibit differences in their resistance to heat: Bacterial spores require >100 °C for destruction Most bacterial vegetative cells are killed at 60-70 °C in 10 min Fungi – 50-60 °C in 10 min Fungal spores – 70-80 °C in 10 min Moist heat can sterilize or disinfect Dry heat Hot air ovens – materials are exposed to high temperatures for a period of time: 150 °C for 90 minutes 160 °C for 60 minutes 170 °C for 40 minutes 180 °C for 20 minutes Used for metallic instruments (like forceps, scissors), glassware (Petri dishes, glass tubes and others). Flaming (burning) – exposure of metallic objects (or other objects that can withstand the temperature) to the flame (300-700 °C) for some time where the flame burns microbes and dust present on the instruments Used for metallic inoculation loops, points of scissors, forceps, rims of test tubes, flasks etc. Moist heat – pasteurization Exposes thermolabile products (milk, wine, beer, others) for a given period of time to a temperature that is high enough to destroy pathogens and spoilage-causing organisms, without necessarily destroying all vegetative cells. High temperature, short time procedure: 71 °C for 15 s. Low temperature, long time procedure: 63 °C for 30 min. Ultra high temperature procedure: 138 °C for 2 s. Rapidly cooled after pasteurization to discourage growth of any remaining viable organisms In microbiology other fluids like vaccines with nonsporing bacteria are pasteurized at 60 °C for 1 hour in special water baths Similarly serum and other body fluids with congeable proteins are sterilized at 56 °C for 1 hour Moist heat – boiling Boiling water or steam at 100 °C is still widely used in veterinary practices (for example, to prepare instruments for a minor surgery). This process kills vegetative bacterial cells and viruses in 30 minutes. Many spores are also killed at 100 °C but some resistant spores for example Clastridium species or Bacillus species can survive boiling for several hours Moist heat – tyndallization Tyndallization (intermittent or fractional sterilization) – the material is exposed to free-flowing steam at 100 °C for 20 minutes on 3 consecutive days with intermittent incubation at 37 °C. Steam kills the vegetative cells → any spores present germinate during incubation time → steam kills the vegetative cells →... Used for materials that are composed of thermolabile chemicals, media containing egg yolk and serum. Replaces by filtration Moist heat – autoclaving Autoclaving – free-flowing steam under pressure, which requires a special piece of equipment – an autoclave. (double walled metal vessel that allows steam to be pressurized in the outer jacket) A pressure of 1.5 atm achieves a temperature of 121 °C and sterilizes materials in 15 minutes. (depends on the heat sensitivity of the materials) Both bacterial vegetative cells and endospores are destroyed. Autoclave is an essential piece of equipment in microbiology laboratories and in operating rooms. At a designated pressure the saturated steam is released into the inner chamber from which all the air has evacuated The steam under pressure in the vacuum inner chamber is now capable of achieving temperatures of 100 °C Used for instruments, dressings, solutions, surgical packs, available for clinicules It is used to sterilize: Different media Different solutions Contaminated materials Used bacterial cultures Different laboratory equipment an other materials Radiation Radiation has various effects on cells depending on it’s wavelength intensity and duration Radiation that kills microorganisms: Non-ionizing – low energy, poor penetrative power (UV light) (has a longer wavelength greater than 1 nm) Ionizing – hight energy, good penetrative powers (X rays, cathode, gamma rays). UV light UV light damages the DNA of exposed cells by causing bonds to form between adjacent pyrimidine bases (usually thymines) in DNA chains → correct DNA replication (during reproduction) is inhibited → bacterial cells are killed UV wavelengths most effective for killing m/o are about 260nm. UV lamps are widely used for surface disinfection in biosafety cabinets, operating theaters and animal quarters in other areas to reduce airborne infections. Major disadvantages – UV light has poor penetrating abilities, so m/o must be directly exposed to the rays. (organisms protected by solutes, paper, glass, textiles are not affected) Antimicrobial activity is also affected by the distance from the UV source. Damaging to humans. (human eyes, burns, skin cancer) Ionizing Radiation Gamma rays, X rays and high-energy electron beams – wavelength shorter than 1 nm. Principal effect – ionization of water → highly reactive hydroxyl radicals → reaction with organic cellular components, especially DNA → cell death. Good penetrative powers – sterilization is achieved in a few seconds. Used to sterilize: Pharmaceuticals Plastic syrignes Surgical gloves Suturing materials Catheters and other heat sensitive materials. Filtration Filtration is the passage of a liquid or gas through a screenlike material with pores small enough to retain m/o. Membrane filters made from cellulose esters and plastic polymers have become popular in recent years. Pore size of 0.45 μm or less removes most or all bacteria from solutions. (cannot remove viruses or mycoplasma) Used to sterilize: Bacteriological media Serum Vaccines Antibiotic solutions and other heat sensitive materials. HEPA filters High efficiency particulate air filters (with porosity of about 0.3 μm) – used to filter air in laboratory rooms, biosafety cabinets and operating theaters. (commonly used when aerosols of contaminated or when sterile surgical conditions are required) 6. Chemical methods of microbial control Principles of effective disinfection Read the label! – indicates what groups of organisms the disinfectant is effective against. Use the correct concentration. (always diluted exactly as said) Consider the environmental pH and temperature – has effect on disinfectant’s activity. Mechanical removal of material → rinsing/washing the surface → once it’s dry – disinfection can be done. Halogens Halogens (particularly iodine and chlorine) are effective antimicrobial agents. Iodine (I2) – impairs protein synthesis and alters cell membranes by forming complexes with amino acids and unsaturated fatty acids. (effective fungi, endospores, viruses) Is available as a: Tincture – a solution in aqueous alcohol Iodophor – combination of iodine and organic molecule, from which the iodine is released slowly. (less irritating, don’t stain) Most common commercial preparation is Betadine (povidone-iodine). (povidone surface active iodophor which improves the wetting action and serves as a reservoir of free iodine) Iodines are mainly used for skin disinfection and wound treatment. Chlorine (Cl2) – used as a gas or in combination with other chemicals. Forms hypochlorous acid (HOCl) with water – a strong oxidizing agent that prevents the function of cellular enzyme system from functioning (most effective form of chlorine, neutral of electrical charge and diffuses as rapidly as water through the cell wall, can be used as aerosol, liquid spray or wet vipes) Compressed chlorine gas – used extensively for disinfecting municipal drinking water, water in swimming pools, and sewage. Calcium hypochlorite Ca(OCl)2) – used in solutions to disinfect, for example, dairy equipment and restaurant eating utensils Chlorine compounds: Sodium hypochlorite (NaOCl) – used as a household disinfectant and bleach, as disinfectant in dairies, food-processing establishments and hemodialysis systems. Chloramine (NH2Cl) – used to disinfect drinking water, eating utensils and food manufacturing equipment. (releases chlorine more slowly) More stable and less irritating than hypochlorite or chlorine gas. Phenols Phenol (carbolic acid) is now rarely used as an antiseptic or disinfectant because it irritates the skin and has disagreeable odour. (was used in surgery to disinfect tools and sewages) Often used in throat lozenges (mediacted tablets) for its local anesthetic effect, but has little antimicrobial effect at low concentrations. At concentrations >1% – significant antibacterial effect. Phenolics – exert antimicrobial activity by injuring lipid containing plasma membranes, which results in leakage of cellular contents. Contain a molecule of phenol that has chemically altered it’s irritating qualities and increase it’s antibacterial activities in combination of soap or detergent Remain active in the presence of organic compounds. they are stable, persist for longer periods after application Suitable agents for disinfecting pus, saliva and feces. One of the most frequently used phenolics is a group of chemicals called cresols. A very important cresol is O-phenylphenol – the main ingredient in most formulation of Lysol. Cresols are very good surface disinfectants. Biguanides Have a broad spectrum of activity, primarily affects bacterial cell membranes. Especially effective against G+ bacteria. (but also for G- bacteria, exception of pseuodomonas, not sporocidal but have some activity against enveloped viruses) Best known biguanide is chlorhexidine – frequently used for microbial control on skin and mucous membranes. In combination with a detergent or alcohol – used for surgical hand scrubs and preoperative skin preparation. Alcohols Alcohols – usually denaturate proteins, but can also disrupt membranes and dissolve lipids. (including lipid component of enveloped viruses) Effectivily kill bacteria, fungi, but not endospores and non-enveloped viruses They act and then evaporate rapidly. (leave no residue, for example swiping the skin with alcohol before injection most of the microbial control activity comes from cleaning off dirt, microorganisms and skin oils) Unsatisfactory antiseptics when applied to wounds they cause coagulation of a layer of protein under which bacteria can continue to grow Most commonly used alcohols are ethanol and isopropanol.(often used to enchance other chemical agents) Recommended concentration of ethanol is 70% (60-95%). Pure ethanol is less effective than aqueous solutions. (denaturation requires water) Isopropanol sold as rubbing alcohol slightly more superior than ethanol as an antiseptic and disinfectant (less volutile, less expensive, more easily obtained than ethanol) Detergents and surfactants Detergents – cleaning agents that contain surfactants (such as soap). Soap has little value as antiseptic, but it helps in the mechanical removal of microbes through scrubbing – good degerming agents. (the skin normally contains dead cells, dust, microbes, dried sweat, oily secretions from sebaceous glands; soap breaks down the oil into tiny droplets it’s called emulsification; water & soap together lift up the emulsified oil and debris and float them away as the lather is washed off) Based on their molecular charge surfactants can be anionic or cationic (or amphoteric, or nonionic). Surfactants - substances that can decrease surface tension among molecules of a liquid Cationic surfactants – quaternary ammonium compounds (quats). Cleansing ability is related to the positively charged portion (cation) of the molecule. Quats affect the plasma membrane, change the cell’s permeability and cause the loss of essential cytoplasmic constituents. (name is derived from the fact that they are modifications of the four balanced ammonium ion) Strongly bactericidal against gram-positive bacteria, less active against gram-negative bacteria. Quats are also fungicidal, amebicidal and virucidal (against enveloped viruses). Inactive against endospores, mycobacteria and certain other bacteria (for example, Pseudomonas which actively grows in quats). Popular quats – benzalkonium chloride and cetylpyridinium chloride. (stronly antimicrobial, colourless, odourless, tasteless, easily diluted, not toxic except at high concentrations, they are used in mouthwashes Aldehydes Effective antimicrobials. Two examples – formaldehyde and glutaraldehyde – inactivate proteins by forming covalent cross-links with several organic functional groups on proteins (-NH2; -OH; -COOH; -SH). Formaldehyde – in gas form it’s an excellent disinfectant, but it is more commonly available as formalin (37% aqueous solution of formaldehyde gas). (EU has banned the use bcs of it’s carcinogenic properties) Formalin was once used extensively to preserve biological specimens and inactivate bacteria and viruses in vaccines. Glutaraldehyde – less irritating and more effective than formaldehyde. Used to disinfect hospital instruments, including endoscopes and respiratory therapy equipment. When used in 2% solution, glutaraldehyde is bactericidal, tuberculocidal, and virucidal in 10 minutes and sporicidal in 3-10 hours. Sterilizing agent! Ethylene oxide gas Gaseous chemosterilant. The application requires a closed chamber similar to steam autoclave. Its activity depends on alkylation – proteins’ labile hydrogen atoms in chemical group (-SH; -COOH; -CH2CH2OH) are replaced with a chemical radicals → cross-linking of nucleic acids and proteins → vital cellular functions are inhibited. The gas kills all microbes and endospores. (but requires many hours) Carries out sterilization at ambient temperatures and is highly penetrating. It is toxic and explosive in its pure form → mixed with nonflammable gas.(CO2) Carries out all sterilisation at ambient temperatures and is highly penetrating (larger hospitals can sterilise matresses with this) Used for instruments that cannot tolerate heat, moisture or abrasive chemicals (such as electronics, optical equipment, paper, rubber and plastics). Ethylene oxide is carcinogenic (group 1), mutagenic, irritating and anaesthetic. Hydrogen Peroxide Peroxygens – a group of oxidizing agents that includes hydrogen peroxide, peracetic acid, ozone and others. (not a good antiseptic for open wounds bcs quickly broken down to water and gaseous oxygen in the action of enzyme catalase which is present in animal cells) H2O2 has the capacity to generate more reactive and cytotoxic oxygen species in the cell, which are powerful oxidants and initiate oxidation of biomolecules. Catalase breaks down H2O2 into water and oxygen, but bacterial enzymes get overwhelmed when high concentrations are used. It effectively disinfects inanimate objects. High concentrations are sporicidal. In a non living surface the normally protective enzymes of aerobic bacteria and facultative anaerobes are ovewhelmed by high concentration of H2O2 , capacity to generate more reactive and cytotoxic oxygen species such as dehydroxyl radical which is a powerful oxidant and iniate oxidation of biomoelcules Microbial characteristics and microbial control Gram-negative bacteria are generally more resistant than Gram-positive bacteria to disinfectants and antiseptics. A principal factor in this relative resistance to biocides is the external lipopolysaccharide layer of Gram-negative bacteria. Pseudomonas and Burkholderia are unusually resistant to biocides, also resistant to many antibiotics. It is mostly related to the characteristics of their porins. (structural openings in cell wall, highly selective of molecules that they permit to enter the cell) Mycobacteria, endospores, and protozoan cysts and oocysts are very resistant to disinfectants and antiseptics. Special tuberculocidal tests have been developed to evaluate the effectiveness of biocides against this bacterial group. Bacterial endospores are affected by relatively few biocides. The cell wall of Mycobacterium spp. have a waxy, lipid-rich component. The resistance of viruses to biocides largely depends on their structure: Antimicrobials that are lipid-soluble are more likely to be effective against enveloped viruses. Nonenveloped viruses are generally more resistant. (protein coat) Prions are resistant to disinfection and autoclaving. (transmissable spongiform encephalopathies, Mad cow disease) WHO and CDC recommends the combined use of a solution of sodium hydroxide and autoclaving at 121 °C for 1 hour or 134 °C for 18 min.