SYLLABUS BASED NOTES FOR THE POST OF JUNIOR LABORATORY ASSISTANT IN MEDICAL EDUCATION PART 1 MICROBIOLOGY (Module 4)

SYLLABUS BASED NOTES FOR THE POST OF JUNIOR LABORATORY ASSISTANT IN MEDICAL EDUCATION PART 1 MICROBIOLOGY (Module 4)

 MODULE IV- 12 Marks

MICROBIOLOGY – 5 Marks

✅ Classification of bacteria based on Morphology.

✅Sterilization methods.

✅ Hot Air Oven, Autoclave (Operation procedure , preparation of articles ,precautions to be taken and use)

✅ Disinfectants & Antiseptics (Application)

• Culture Media: Classification of culture media with example ( knowledge assessment)

• Culture Methods: Streak, Stroke, Stab, Lawn culture, Anaerobic technique (gaspak) (Knowledge assessment)


•Father of microbiology -LOUIS PASTEUR 

•Father of antiseptic surgery -JOSEPH LISTER 

•Father of bacteriology -ROBERT KOCH

•Father of immunology -EDWARD JENNER 

•Father of chemotherapy -PAUL EHRLICH

INTRODUCTION 

•Bacteria are unicellular
• Possess both DNA and RNA 
• Prokaryotic microoraganisms
•Occur in water, soil, air, foof, and all natural environment 
•Survive extremes of temperature, pH, oxygen, etc... 
•Devoid of chlorophyll

CLASSIFICATION 

Depending upon their shape, bacteria are classified into; 

1.COCCI 
•They are spherical in shape . 
•They are: MONOCOCCUS [single] 
DIPLOCOCCUS [ two ] 
TETRACOCCUS [ four ] - TETRAD •STREPTOCOCCUS [ chains ] 
SARCINA [eight] 
STAPHYLOCOCCUS [ Cluster]




2.BACILLI 
•Cylindrical or rod shaped 
COCCOBACILLIUS [brucella ] 
DIPLOBACILLI 
PALISADES 
STREPTOBACILLI 
CHINESE LETTER OR CUNEIFORM PATTERN
 [ corynebacterium diphtheriae]




3. VIBRIO 
Comma or curved shaped 
Shows vibratory motility 

4.SPIROCHETES 
Thin spirally coiled 

5.ACTINOMYCETES [ sun ray appearance] 
They are branching filamentous bacteria 
Resumble to radiating sun rays. 

6.MYCOPLASMA 
Lack cell wall 
Donot possess a stable morphology


STERILIZATION 

Process by which all the microorganisms which include viable spores may either destroyed or removed from an article, body surface or medium 

DISINFECTION 
Process that destroy or removes most if not all the pathogenic organisms but not bacterial spores. 

ASEPSIS 
Process where the chemical agents are applied on the body surfaces which kill or inhibit the microorganism present on the skin

1.DRY HEAT 

RED HEAT 
•Rapid sterilization method is the direct flame method
•Inoculating loops and wires, points of forceps are held in a bunsen flame till it become red hot for sterlization 

FLAMING 
•Passed a few times through the bunsen flame without allowing them to red hot 
• Needles, mouth of the culture tubes, glass slide, cover slip 

INCINERATION
• Used for the disposal of biomedical waste materials 
• Burns very high temperature [ 870-1200 degree C] and convert the waste into ash, flue gas heat

HOT AIR OVEN [ DRY HEAT STERILIZER] 

•Most widely used method of sterilization by dry heat •It is a metal cabinet with three walls and an interlocking door 
•It is electrically heated and fitted with a fan to even distribution of hot air in the chamber 
•A thermostat is fitted which maintain the chose temperature in chamber 
• Temperature - 160 degree C for 2 hour

MATERIALS STERILIZED 
•Glassware like glass syringes, petri dishes, flasks, pipettes and test tubes 
•Surgical instruments like scalpels, forceps, 
•Chemicals such as liquid paraffin, fats, glycerol and glove powder, etc

PRECAUTIONS 
• Materials should not be overloaded 
• Materials should be arranged in a manner 
• Glass wares should be perfectly dry before placed in the oven. 
•Test tubes, flasks, should be plugged with cotton wool
•Liquid and culture media should not be placed

STERILIZATION CONTROL 
Biological indicator - spores of bacillus atrophaeus are used to check the effectiveness of sterilization by dr heat.
 [ earlier non toxigenic strains of clostridium tetani used]

2.MOIST HEAT 
Temperature below 100 degree C 

1)PASTEURIZATION
 • Used to control microorganism from beverages like fruits, juices and dairy products such as milk 
• 2 methods are available
*HOLDER METHOD [ 63 degreeC for 30 minute]
*FLASH METHOD [ 72 degreeC for 20 seconds] followed by rapid cooling to 13 degree or lower
•All nonsporing pathogens including mycobacte brucella are killed except coxiella burnetti which relatively heat resistant

2) WATER BATH 
• Used to disinfection of serum, body fluid and vaccines 
• Bacterial vaccines are disinfected at 60 degreeC for 1hr. 
•Serum or heat labile body fluid can be disinfected at 56 degreeC for 1 hr. 

3) INSPISSATION [ fractional sterilization] 
• Process of heating an article on 3 successive days at 80-85 degreeC for 30 minutes by a special instrument called inspissator

WORKING PRINCIPLE
 • The first exposure kills all the vegetative forms, in between interval spores germinate into vegeta forms and killed on subsequent killing

MOIST HEAT AT 100 DEGREE C 
•Boiling 
• Steaming 
•Tyndallization 

MOIST HEAT ABOVE 100 DEGREE C[ autoaclave ] 
Similar to pressure cooker Water boils when vapour pressure is equal that of the surrounding atmosphere. . So when the atmospheric pressure raised, the boiling temperature is also raised the boiling temperature is also raised. 
• At normal pressure water boils at 100 degree C but when pressure inside a closed vessel increases the temperature at which water oils also increases

COMPONENTS 
• Pressure chamber A lid 
• An electric heater pressure chamber consist of; 
Large cylinder in which materials to be sterilized are . placed. . 
Made up of gunmetal or staunless steel placed in a supporting iron case. 
• A steam jacket [water compartment] 

Lid consist of: 
• A discharge tap for air and steam discharge . 
•A pressure gauge [ set pressure level] 
•A safety valve [remove the excess steam ]
•A electrical heater is attached to the jacket; that heats the water to produce steam. 



PROCEDURE 
•Cylinder is filled with sufficient water and materials placed inside the pressure chamber. 
•Lid is closed and electric heater is put on Safety valve is adjusted to the required pessure 
• Water boils steam and air mixture is allowed to escape through the discharge tap till all the air has been displaced.
 [this is tested by passing the steam air mixture liberated ffrom the discharge tap in to a pail of water air bubbles stop comig in the pail indicates air has been displaced by steam ]
• The steam pressure rises inside and when it reaches the desired level, the safety valve is opens and excess steam escapes out. 
•Holding period is counted from this point which is about 15 minutes in most cases. 
• After holding period, the electric heater is stopped and autoclave is allowed to cool till the pressure gauge indicates pressure inside is equal to atmospheric pressure. 
•Discharge tap is opened slowly and air allowed to enter in the autoclave. 
•Lid now opened and materials are removed. 

STERILIZATION CONDITIONS 
•121 degree C for 15 minutes at pressure 15lbs 
•126 degree C for 10 minutes at pressure 20lbs 
• 133 degree C for 3 minutes at pressure 30 lbs

USES 
Method of choice of sterilizing:
• Surgical instruments 
• Culture media 
•Solutions and water Biomedical waste 
• Glassware 

PRECAUTIONS 
•Should not be used for sterilizing waterprood materia 
• Donot over fill the chamber 
• Materials should not touch the sides or top of the chamber 
•Clean item and wastes should be autoclaved seperately

CHEMICAL METHODS OF DISINFECTION 
Disinfectants are those chemicals that destroy pathogenic bacteria from inanimate surfaces. 
Some chemicals when used at apropriate concentration for appropriate duration can be used for sterilization and are called sterilant liquids

Those chemicals that can be safely applied over skin and mucus membranes are called antiseptics

An ideal antiseptic or disinfectant should have following properties: Notes 
1. Should have wide spectrum of activity 
2. Should be able to destroy microbes within practical period of time 
3. Should be active in the presence of organic matter 
4. Should make effective contact and be wettable 
5. Should be active in any pH 
6. Should be stable 
7. Should have long shelf life 
8. Should be speedy
9.Should have high penetrating power 
10-Should be non-toxic, non-allergenic, non-irritative or non-corrosive . 
11.Should not have bad odour 
12.Should not leave non-volatile residue or stain 
13. Efficacy should not be lost on reasonable dilution 14. Should not be expensive and must be available easily

Classifications of disinfectants:
 1. Based on consistency
(a) Liquid (Eg, Alcohols, Phenols) 
(b) Gaseous (Formaldehyde vapour) 

2. Based on spectrum of activity 
(a) High level 
(b) Intermediate level 
(c) Low level

Alcohols 

Mode of action: 
Alcohols dehydrate cells, disrupt membranes and cause coagulation of protein. 
Examples: Ethyl alcohol, isopropyl alcohol and methyl alcohol 

Application:
 A 70% aqueous solution is more effective at killing microbes than absolute alcohols. 
70% ethyl alcohol (spirit) is used as antiseptic on skin. 

Isopropyl alcohol is preferred to ethanol. 
It can also be used to disinfect surfaces. It is used to disinfect clinical thermometers. 
Methyl alcohol kills fungal spores, hence is useful in disinfecting inoculation hoods. 

Disadvantages: Skin irritant, volatile (evaporates rapidly), inflammable 

Aldehydes
Mode of action: Acts through alkylation of amino-, carboxyl- or hydroxyl group. and probably damages nucleicacids. It kills all microorganisms, including spores. 

Examples: Formaldehyde, Gluteraldehyde 

Application
40% Formaldehyde (formalin) is used for surface disinfection and fumigation of rooms, chambers, operation theatres, biological safety cabinets, wards, sick rooms etc. 
Fumigation is achieved by boiling formalin, heating paraformaldehyde or treating formalin with potassium permanganate. 
It also sterilizes bedding, furniture and books. 10% formalin with 0.5% tetraborate sterilizes clean metal instruments. 2% gluteraldehyde is used to sterilize thermometers, cystoscopes, bronchoscopes, centrifuges, anasethetic equipments etc. 
An exposure of at least 3 hours at alkaline pH is required for action by gluteraldehyde. 2% formaldehyde at 40°C for 20 minutes is used to disinfect wool and 0.25% at 60°C for six hours to disinfect animal hair and bristles

Phenol 
Mode of action: 
Act by disruption of membranes, precipitation of proteins and inactivation of enzymes. Notes 

Examples: 5% phenol, 1-5% Cresol, 5% Lysol (a saponified cresol). hexachlorophene, chlorhexidine, chloroxylenol (Dettol) 

Applications
Joseph Lister used it to prevent infection of surgical wounds. Phenols are coal-tar derivatives. They act as disinfectants at high concentration and as antiseptics at low concentrations. They are bactericidal, fungicidal, mycobactericidal but are inactive against spores and most viruses. They are not readily inactivated by organic matter. The corrosive phenolics are used for disinfection of ward floors, in discarding jars in laboratories and disinfection of bedpans. Chlorhexidine can be used in an isopropanol solution for skin disinfection, or as an aqueous solution for wound irrigation. It is often used as an antiseptic hand wash. 

Disadvantages: It is toxic, corrosive and skin irritant. Chlorhexidine is inactivated by anionic soaps. Chloroxylenol is inactivated by hard water. 

Halogens 
Modes of action: They are oxidizing agents and cause damage by oxidation of essential sulfydryl groups of enzymes. Chlorine reacts with water to form hypochlorous acid, which is microbicidal

Applications: 
Tincture of iodine (2% iodine in 70% alcohol) is an antiseptic. Iodine can be combined with neutral carrier polymers such as polyvinylpyrrolidone to prepare iodophores such as povidone-iodine. Iodophores permit slow release and reduce the irritation of the antiseptic. For hand washing iodophores are diluted in 50% alcohol. 10% Povidone Iodine is used undiluted in pre and postoperative skin disinfection. Chlorine gas is used to bleach water. Household bleach can be used to disinfect floors. Household bleach used in a stock dilution of 1:10. In higher concentrations chlorine is used to disinfect swimming pools. 0.5% sodium hypochlorite is used in serology and virology. Used at a dilution of 1:10 in decontamination of spillage of infectious material. Mercuric chloride is used as a disinfectant. Notes 

Disadvantages: They are rapidly inactivated in the presence of organic matter. Iodine is corrosive and staining Bleach solution is corrosive and will corrode stainless steel surfaces

Heavy Metals 
Mode of action: 
Act by precipitation of proteins and oxidation of sulfydryl groups. They are bacteriostatic.

 Examples: Mercuric chloride, silver nitrate, copper sulfate, organic mercury salts (eg, mercurochrome, merthiolate) 

Applications: 1% silver nitrate solution can be applied on eyes as treatment for opthalmia neonatorum (Crede's method). This procedure is no longer followed. Silver sulphadiazine is used topically to help to prevent colonization and infection of burn tissues. Mercurials are active against viruses at dilution of 1:500 to 1:1000. Merthiolate at a concentration of 1:10000 is used in preservation of serum. Copper salts are used as a fungicide

Surface Active Agents Mode of actions: 
They have the property of concentrating at interfaces between lipid containing membrane of bacterial cell and surrounding aqueous medium. 
These compounds have long chain hydrocarbons that are fat soluble and charged ions that are water-soluble. Since they contain both of these, they concentrate on the surface of membranes. 
They disrupt membrane resulting in leakage of cell constituents made to have a positive charge by combining with a quaternary nitrogen atom, it is called cationic detergents. Cationic detergents are known as quaternary ammonium compounds for quat) Cetrimide and benzalkonium chloride act as cationic detergents. 

Application: They are active against vegetative cells, Mycobacteria and enveloped viruses. They are widely used as disinfectants at dilution of 1-2% for domestic use and in hospitals. Notes Disadvantages: Their activity is reduced by hard water, anionic detergents and organic matter. Pseudomonas can metabolise cetrimide, using them as a carbon, nitrogen and energy source. 

Dyes 
Mode of action: Acridine dyes are bactericidal because of their interaction with bacterial nucleic acids

Acridine dyes such as acriflavin and aminacrine. Acriflavine is a mixture of proflavine and cuflavine. Only cuflavine has effective antimicrobial properties. They are more effective against gram positive bacteria than gram negative bacteria and are more bacteriostatic in action. 

Applications: They may be used topically as antiseptics to treat mild burns. They are used as paint on the skin to treat bacterial skin infections. Melachite green is used in LJ medium for growth of Mycobacterium tuberculosis. 

Hydrogen Peroxide 
Mode of action: It acts on the microorganisms through its release of nascent oxygen. Hydrogen peroxide produces hydroxyl-free radical that damages proteins and DNA. 

Application: It is used at 6% concentration to decontaminate the instruments, equipments such as ventilators. 3% Hydrogen Peroxide Solution is used for skin disinfection and deodorising wounds and ulcers. Strong solutions are sporicidal. 

Disadvantages: Decomposes in light, broken down by catalase, proteinaceous organic matter drastically reduces its activity


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