General Microbiology

Historical Introduction Important Notes

1. Scientists and their contribution

2. Staining

Historical Introduction Short Answers

Question 1. Koch’s postulates/Robert Koch?
Answer:

. Koch’s postulates/Robert Koch

• Robert Koch is known as the father of medical microbiology.
• His contributions to microbiology are:

1. Robert Koch Introduced

• Methods of obtaining bacteria in pure cultures using solid media.
• Staining techniques for bacteria
• Methods for isolation of pure strains of bacteria.

2. Robert Koch Discovered

• Anthrax bacillus
• Tubercle bacillus
• Cholera vibrios.

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3. Robert Koch Proposed Koch’s Postulates

• Koch’s postulates:
  • According to Koch’s postulates, a micro-organism can be accepted as the causative agent of an infectious disease only if
    • The organism is constantly associated with the lesions of the disease
    • If it is possible to isolate the organism in pure cultures from the lesions of the disease.
    • Inoculation of such pure culture into suitable laboratory animals will reproduce the lesions of the disease.
    • If it is possible to re-isolate the bacterium in pure culture from the lesions produced in the experimental animals.

4. He demonstrated Koch’s phenomenon through his observation of guinea pigs

• It is as follows
  • Koch’s phenomenon:
    • Robert Koch observed that guinea pigs already infected with tubercle bacillus responded with an exaggerated inflammatory response when injected with the tubercle bacillus (or) its protein.
    • This hypersensitivity reaction is known as Koch’s phenomenon.

Question 2. Louis Pasteur.
Answer:

Louis Pasteur

• He was a trained chemist in France.
• He is known as the father of microbiology.

Important contributions in microbiology:

• He developed methods and techniques of bacteriology.
• Introduced sterilization techniques.
• Developed steam sterilizer, autoclave, and hot air oven
• Developed live attenuated vaccine for prophylactic use
• He studied
  • Chicken cholera
  • Anthrax
  • Hydrophobia
• He also coined the term vaccine.
• He proved that all forms of life even microbes arose only from their like and not de novo.
• He proposed a microbial theory of fermentation to explain the role of microorganisms in disease production
• He controlled the diseases of silkworm

Morphology And Physiology Of Bacteria Important Notes

1. Bacteria and their arrangement

2. Contents of bacterial cell

• Cell
• Cytoplasmic membrane
• Cytoplasm
• Nucleus
• Capsule
• Flagella
• Fimbriae
• Spore

3. Organism and shape of its spores

Morphology And Physiology Of Bacteria Long Essays

Question 1. Classify bacteria depending on their shape. 
Answer:

Morphology And Physiology Of Bacteria

Depending upon the shape, bacteria are classified into:

Morphology And Physiology Of Bacteria Short Essays

Question 1. Bacterial Spore
Answer:

Bacterial Spore

• Spores are highly resistant resting stages of the bacteria formed in unfavorable environmental conditions.
• Sporulation is not a method of reproduction.

Morphology of Spore

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Spores consist of the following structures Cell

• Some of the spores contain exosporium
• It is the loose outer covering

Morphology And Physiology Of Bacteria

Bacterial Spore Shape and Position:

• Bacterial Spore Position
  • Spores may be
    • Central
    • Subterminal (or)
    • Terminal
• Bacterial Spore Shape:
  • They may be
    • Spherical
    • Oval
      • Depending on the diameter, they may be
    • Bulging
    • Non-bulging.

Bacterial Spore Properties:

• Resistance
  • Spores are extremely resistant to ordinary boiling, disinfectants, and heating.
• Used for sterilization
  • Spores of some species of bacteria act as indicators for proper sterilization
• Gemination
  • Gemination is the process of spore conversion into vegetative cells under favorable conditions.

Bacterial Spore Demonstration:

Spore Forming Bacteria are:

Question 2. Gram staining/Gram stain?
Answer:

Gram staining/Gram stain

• Gram stain is the most widely used stain in bacteriology.
• Gram staining is the essential procedure used in the identification of bacteria and is frequently the only method required to study their morphology.

Gram staining Method:

• Gram staining involves four basic steps.
  • The primary staining with a pararosaniline dye such as crystal violet, methyl violet (or) gentian violet for one minute
  • Application of gram’s iodine [dilute solution of io¬dine) over the slide for one minute.
  • Decolorization with an organic solvent such as ethanol, acetone (or) aniline for 10-30 seconds
  • Counterstaining with a dye of contrasting color such as carbon fuchsin, safranin, or neutral red for 30 seconds

Differentiation on gram staining: It is called a differential stain because it differentiates between gram-positive and gram-negative bacteria

Gram staining Mechanism:

Morphology And Physiology Of Bacteria

1. The exact mechanism is not understood.

2. It may be attributed to the following:

• Permeability of bacterial cell wall and cytoplasmic membrane:
  • The protoplasm of the Gram-positive cells is more acidic than that of Gram-negative cell
  • Due to this, it retains the basic dyes strongly
  • Now when iodine is added to it, the acidic nature of the protoplasm increases
  • As a result, iodine combines with the dye and forms a dye-iodine complex and fixes the dye in the bacterial cell
  • The Cytoplasmic membrane of Gram-positive cells is less permeable to this complex
  • Thus the dye-iodine complex gets trapped within the cell
  • In contrast, the Gram-negative cell wall has increased permeability
  • This leads to the outflow of the complex during decolorization
• The integrity of the cell wall
  • If the cell wall is damaged, the Gram-positive bacteria becomes Gram-negative

Question 3. Acid fast stain/Ziehl Neelsan stain?
Answer:

Acid fast stain/Ziehl Neelsan stain

• The acid-fast stain was discovered by Ehrlich and subsequently modified by Ziehl and Neelsen.
• Some organism retains carbol fuchsin even when colorized with acid. Such organisms are called acid-fast organisms.
• Example: Mycobacteria

Acid-fast stain Method:

• Pour carbol fuchsin satin on a slide containing a fixed smear
• Gently heat the underside of the slide till it produces steam
• Leave carbol fuchsin over the slide for 5-10 minutes along with intermittent heating
• Don’t allow the slide to dry out for it adds stain to it and reheat
• Wash the slide in tap water
• Decolorize the smear with 20% sulphuric acid and wash it With water
• Repeat the procedure till the pink/ red color stops coming out
• Counter-stain the smear with 2% methylene blue for 1¬2 minute
• Wash it with water and air dry it
• Observe under microscope

Acid-fast stain Microscopic Examination: Acid-fast bacilli appear red in the blue background of pushing cells and epithelial cells

Acid-fast stain Principle:

• Acid fastness depends on
• The high content of lipids, fatty acids, and higher alcohols found in the cell wall of Mycobacterium
• The integrity of the cell wall

Question 4. Name four staining techniques in microbiology.
Answer:

Commonly used staining techniques in microbiology are as follows.

Question 5. Bacterial cell wall
Answer:

Bacterial cell wall

• The cell wall is a tough and rigid structure
• It surrounds the bacteria like a shell

Bacterial cell wall Functions:

• Accounts for the shape of the cell
• It takes part in cell division
• Protects the cell against osmotic damage
• Provide rigidity
• It possesses a target site for antibiotics, lysosomes, and bacteriophages

Morphology And Physiology Of Bacteria

Bacterial cell wall Structure:

• The rigid part of the cell wall is peptidoglycan
• It has the following components

1. Lipoprotein layer It connects the peptidoglycan to the outer membrane

2. Outer membrane

• It contains certain proteins named outer merm brane proteins
• These are target sites for bacteriocins

3. Lipo-polysacharride .

• This layer consists of lipid A to which polysaccha¬ride is attached
• The toxicity is associated with lipid A
• The polysaccharide determines a major surface an¬tigen to 0 antigen
• It contains endotoxin in gram-negative bacteria
• It is composed of 3 regions as follows
  • Region 1 – polysaccharide portion (0 antigen specificity)
  • Region 2 – core polysaccharide
  • Region 3 – Lipid A portion (responsible for tox¬icity)

4. Periplasmic space

• It is the space between the inner and outer membrane
• It contains various binding proteins

5. Peptidoglycan

Morphology And Physiology Of Bacteria Short Question And Answers

Question 1. Name capsulated bacteria
Answer:

Name capsulated bacteria

• Streptococcus pneumonia
• Klebsiella sp.
• Bacillus antacids
• Cryptococcus neoformans. (a fungus)

Question 2. Fimbriae
Answer:

Fimbriae

• Fimbriae is also called pili
• These are hair-like appendages projecting from the cell surface as straight filaments.

Fimbriae Types:

• Common pili
• Sex of F (fertility) pili
• Col 1 (colicin) pili

Fimbriae Functions:

• Adhesion
• Transfer of genetic material

Morphology And Physiology Of Bacteria

Fimbriae Demonstration:

• Fimbriae are demonstrated by
  • Electron microscopy
  • Haemagglutination test

Question 3. Flagella
Answer:

Flagella

Flagella are cytoplasmic appendages protruding through a cell wall.

Flagella Structure:

• They are thread-like structures
• Size:
  • Length – 5-20 micrometers
  • Diameter- 0.01-0.02 micrometer

Flagella Parts:

• It is composed of three parts
  • Filament
    • It lies external to the cell
    • It is connected to the hook at the cell surface ‘250
  • Hook
    • The hook-basal body is embedded in the cell envelope
  • Basal body.
    • It contains outer and inner rings

Flagella Composition:

• The flagella is made up of flagellin, a protein
• Specific flagellar antibodies are produced in high titers

Flagella Functions:

• These are organs of locomotion.
• Flagellar antibodies are used for serodiagnosis

Flagella Types:

Question 4. Differences between Gram-positive and Gram-negative cell walls:
Answer:

Differences between Gram-positive and Gram-negative cell walls

Morphology And Physiology Of Bacteria Viva Voce

1. Gram-positive bacteria appear violet and Gram-negative bacteria appear red on staining
2. In acid-fast staining, a positive reaction gives a red color while a negative reaction gives a blue color
3. All cocci except Neisseria are Gram-positive
4. Flagella is a locomotory organ
5. The nucleus of bacteria consists of plasmids or episomes
6. Bacteria without cell walls are called Mycoplasma

Infection Important Notes

1. Types of infection

2. Sources of infection

3. Exotoxin

• Generally formed by Gram-positive bacteria
• Heat labile, highly antigenic, highly specific
• Inactivated by formalin
• Converts toxins into toxoids

4. Endotoxin

• Produced by Gram-negative bacteria
• Heat stable, less potent, nonspecific
• Cannot be toxoided

Infection Short Essays

Question 1. Modes of transmission of infection.
Answer:

Modes of transmission of infection

1. Contact:

• It may be direct (or) indirect
• Infections spread by direct contact are sexually transmitted diseases such as syphilis, AIDS, and gonorrhea.
• A contagious disease is a term used for the disease acquired by direct contact
• Indirect contact may be through the agency of fomites which are inanimate objects such as clothing, toys, etc.

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2. Inhalation:

• Respiratory infections such as the common cold, influenza, and T.B. are acquired by inhalation.
• These organisms are shed into the environment by patients in secretions from the nose (or) throat during sneezing, coughing (or) speaking.

3. Inoculation: In some instances, pathogens may be directly inoculated into the tissues of the host For Example: Tetanus spores in deep wounds, and rabies virus directly by bite of a rabid animal.

4. Ingestion: Intestinal infections like cholera, dysentery, food poisoning, and most of parasitic infections are acquired by ingestion of food (or) drink contaminated by pathogens.

5. Vectors:

• Vectors are anthropods or other invertebrate hosts Examples: are flies, ticks, and mosquitoes.
• Transmission by vector may be either mechanical or biological.

6. Transplacental transmission: Some pathogens can cross the placental barrier and infect the fetus in utero. This is known as vertical transmission.

Example. Teratogenic infections like rubella.

7. Iatrogenic and laboratory infections:

• If meticulous care in asepsis is lacking certain infections like AIDS and hepatitis B may sometime be transmitted during procedures such as injections, lumbar punctures, blood transfusions, and surgery. These are known as iatrogenic infections.
• Laboratory personnel handling infectious material are at risk of infection transmission.

Question 2. Sources of infection?
Answer:

Sources of infection

Sources of infection may be endogenous (or) exogenous sources.

1. Endogenus sources: Organisms of normal flora are usually non-pathogenic but occasionally they behave as pathogens outside their habitat.

Example: Virtdians streptococci (normal flora of mouth) can cause infective endocarditis.

2. Exogenous sources:

• Most of the infections are exogenous In origin.
• Exogenous sources include.
  • Human cases and carriers.
    • Humans themselves are the most common source of infection.
  • Animals.

• • Insects
    • Blood-sucking insects such as mosquitoes, ticks mites, and flies may transmit several pathogens to man.
    • Anopheles mosquito – malaria.
  • Soil and water
    • Spores of tetanus and gas gangrene bacilli remain viable in the soil for decades and serve as a source of infection.
  • Food:
    • Contaminated food acts as a source of infection in cases of food poisoning, diarrhea, and dysentery.
    • Example: Food poisoning by staphylococcus.

Question 3. Differences between Exotoxin and endotoxin.
Answer:

Differences between Exotoxin and endotoxin

Question 4. Nosocomial or hospital-acquired infections?
Answer:

Nosocomial or hospital-acquired infections

• Cross-infection acquired in hospitals is called hospital-acquired hospital-associated or nosocomial infection.
• They may manifest during their stay in the hospital or sometimes after the patient is discharged.
• They may spread through droplets infection, dust, skin scales, in animate sources.
• Direct spread through contact and indirectly through equipment and materials.

Nosocomial Infections and Causative Organisms

Prevention or Control of Hospital-Acquired Infections:

• Proper sterilization and disinfection of the inanimate objects.
• Control of route of transmission by regular washings of hands, and disinfection of equipment.
• Isolation of an infectious patient
• Administration of antibiotics and antiseptics to the carrier staff (or) source patient
• Vaccination to susceptible hosts.

Bacterial Genetics Important Notes

1. Some important terms

2. Types of drug resistance

• Mutational drug resistance
• Transferable drug resistance

Bacterial Genetics Short Essays

Question 1. Methods of Genetic Transfer
Answer:

Gene transfer in bacteria occurs by following methods

• Genetic Transformation
  • It is the transfer of genetic information through free or naked DNA
  • Factors influencing the transformation are
    • The physical state of donor DNA
    • Competency of the recipient cell
    • The fate of DNA upon entering the cell
  • It occurs naturally in Bacillus subtilis, Streptococcus pneumonia, Hemophilus influenza, Neis erria
  • It can be induced in the laboratory
• Genetic Transfer Transduction
  • It is the transmission of a portion of DNA from one bacterium to another by a bacteriophage
  • A phage particle may carry a segment of host DNA besides its own nucleic acid
  • When this infects another bacterium DNA transfer is affected and the recipient cell acquires new characteristics coded by the donor DNA
  • It is not confined to transfer to chromosomal DNA
  • but can transfer episomes and plasmids
• Genetic Transfer Significance
  • Widespread mechanism
  • Used for genetic mapping
  • Used In genetic engineering In the treatment of some Inborn errors of metabolism
• Lysogenic conversion
  • In this method, a new genetic material Is Incorporated Into the bacterial chromosome known as prophage
  • This carries genetic Information to a bacterium which may code for new characteristics
  • The bacteria possessing this prophage is known as a lysogenic bacterium
• Genetic Transfer Conjugation
  • The transfer of genetic information from one bacterium to another through matting is called conjugation
  • It takes place between a male cell that consists of F plasmid and a female cell which lacks F plasmid
  • The donor DNA combines with the recipient DNA resulting in genetic recombination

Bacterial Genetics Short Question And Answers

Question 1. Transfer factor:
Answer:

Transfer factor

• Transfer factor is a low molecular weight substance resistant to trypsin but gets inactivated at 56°C for 30 minutes.
• It plays a role in the transfer of cell-mediated immune response in man.
• It is not antigenic.
• The transferred cell-mediated immune response is systemic and not local.
• The mechanism of action is not known. But it may stimulate the release of lymphokines from sensitized T lymphocytes.

Transfer factor Uses:

• Used in T cell deficiency (Wiskott Aldrich syndrome) patients.
• Treatment of malignant melanoma and other type of cancer.
• Used in the treatment of tuberculosis and lepromatous leprosy.

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Question 2. DNA probes
Answer:

DNA probes

DNA probes are radiolabelled or chromogenically labeled pieces of single-stranded DNA which can be used for the detection of homologous DNA by hybridization

DNA probes Advantages:

• The high degree of specificity
• Able to detect minute amounts of complementary DNA

DNA probes Use:

• In clinical microbiology
• Direct detection of microbes in specimens
• To detect microbes which are either difficult or impossible to culture
  • Identification of culture isolates
  • Strain identification
  • To identify toxins, virulence factors
  • Identification of resistant markers

Question 3. Plasmids
Answer:

Plasmids

• Plasmids are double-stranded circular DNA molecules present in the cytoplasm of bacteria
• They are capable of autonomous replication
• They are important vectors in genetic engineering
• They are able to transfer genes from one bacterium to another
• Two members of the same group of plasmids cannot occur in the same cells
• They confer properties like toxigenicity and drug resistance

Bacterial Genetics Viva Voce

1. Restriction endonucleases are enzymes that cleave double-stranded DNA
2. Southern blotting identifies DNA fragments
3. Northern blotting analyses RNA
4. Western blotting identifies antigens
5. Pathogenicity is the ability of microbial species to produce disease
6. Virulence is degree of Pathogenicity of microbe

Identification Of Bacteria Important Notes

1. Types of microscopy used for the identification of bacteria

• Optical or light microscopy
• Phase contrast microscopy
• Darkfield microscopy
• Interference microscope
• Polarization microscope
• Electronic microscope

2. Staining of bacteria

Identification Of Bacteria Short Answers

Question 1. Indole test?
Answer:

Indole test Principle: To determine the ability of an organism to decompose amino acid tryptophan into indole.

Indole test Procedure:

• Inoculate the test bacterium into tryptophan-rich peptone water and incubate at 37°C for 48-96 hours.
• 0.5 ml of Kovac’s reagent is added and gently shaken.

Indole test Interpretation:

• Indole positive – A red-coloured ring near the surface of the medium
• Indole negative – A yellow-coloured ring near the surface of the medium.

Indole test Use:

• To identify and classify bacteria.
  • Indole positive: E.Coli, Proteus species
  • Indole negative: Klebsiella species

Question 2. Urease test?
Answer:

Urease test Principle: To determine the ability of an organism to produce an enzyme urease which splits urea to ammonia.

Urease test Procedure:

• The test is done is Christensen’s urease medium
• Test organism was inoculated on the entire slope of the medium and incubated at 37°C.
• It is examined after 4 hours and overnight incubation.

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Urease test Interpretation:

Question 3. Voges – proskouev test:
Answer:

Voges – Proskauer test Principle: This test depends on the production of acetyl methyl- carbinol from pyruvic acid as an intermediate stage in its conversion to 2:3 butylene glycol.

Voges – proskouev test Procedure:

• 1 ml of glucose phosphate medium culture of the organisms incubated at 30°C for 5 days (or) 37°C for 48 hours is taken
• Then 1 volume (0.2 ml) of 40% KOH and 3 volumes (0.6 ml) of 5% a-naphthol solution in ethanol are added.

Voges – proskouev test Interpretation:

• Positive- Pink colour appears in 2 – Minutes
• Negative- No colour change for 30 minutes it remains colourless.

Identification Of Bacteria Viva Voce

1. Casoni test is used to diagnose hydatid cyst
2. DNA in bacteria is composed of two strands of complementary nucleotides wound together
3. Weil Felix reaction is used for typhus fever
4. Paul Bunnel test is used for infectious mononucleosis
5. Montoux test is used for tuberculosis
6. Naggler’s reaction is used for Clostridium

Culture Media And Culture Methods Important Notes

1. Culture media

2. Common culture media used in mycology

• Sabouraud’s agar
• Cornmeal agar

3. Czapek – Dox

• Anaerobic culture method
  • Obtained by McIntosh and Flide’s anaerobic jar
  • It is the most reliable and widely used method

Culture Media And Culture Methods Long Essays

Question 1. Define and classify culture media with examples. Write briefly on selective and anaerobic media.
Answer:

Culture media

The culture media are the bacteriological media used for isolation and characterization of various bacterial pathogens

Cultural media Classification: Cultural media are classified as:

Selective Media:

• They are solid media which contain a substance incorporated into it
• It has a selective stimulating effect on the bacteria to be grown and an inhibitory effect on the growth of the unwanted bacteria
• They enable a greater number of the required bacteria to form colonies
• They are used to isolate a particular bacteria from specimens

Anaerobic Media:

• These media are used to grow the anaerobic organism
• They contain a reducing agent that absorbs all the oxygen and creates an anaerobic condition favouring the growth of anaerobes

Question 2. Classify culture media. Describe anaerobic culture methods.
(or)
McIntosh and Filde’s anaerobic jar
Answer:

Anaerobic Culture Methods:

1. Cultivation in vacuum.

• Anaerobic chamber (or) cabinets (or) glove boxes
• Anaerobic bags (or) pouches

2. Displacement of oxygen with gases like hydrogen, nitrogen, helium (or) CO2. Candle jar method.

3. Absorption of oxygen.

• Chemical methods
  • Alkaline pyrogallol method.
    • Spray anaerobic dish
    • Ordinary petri dish.
  • Buchner’s tube
  • Rosenthal method
  • Phosphoprous stick method.
• Biological methods.

4. Me Intosh filde anaerobic jar

• It is most widely used anaerobic method
• It consists of a glass or metal jar with a metal lid that can be clamped down on a gasket to make it airtight with a screw clamp
• The lid has two tubes with taps, one is gas inlet and the other is an outlet

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• One or more capsules containing palladiumized alumina pellets are suspended under the lid with wires and connected to electric terminals on the lid for heating
• Alternatively a temperature catalyst called de-oxo pellets can be used
• Inoculated culture plates are placed in the jar with the medium in the bottom half of the plates and lids are clamped tight
• The production of anaerobic conditions is brought about by
  • Displacement of oxygen
  • Displacement and combustion of oxygen

5. Gaspak system

6. Reduction of oxygen.

• An aerobic media
  • Example: Robertson’s cooked meat broth
• Thioglycollate medium
• Smith Noguchi medium.

Culture Media And Culture Methods Short Essays

Question 1. Enriched media
Answer:

Enriched media

• This is a special medium,
• When a basal medium is added with some nutrients such as blood, serum (or) egg it is called an enriched medium.
• Enriched media = Basal media + some nutrients [blood, serum, egg]

Enriched media Examples:

• Blood agar:
  • Blood agar – nutrient agar + blood
  • Used for routine culture
  • Example: streptococcus
  • Mostly sheep blood 5 – 10% is used.
• Chocolate agar:
  • It is prepared by heating 10% sterile blood in sterile nutrient agar at 55oC for 2 hours.
  • Used for the cultivation of Neisse via, Haemophilus influenza.
• Loeffler’s serum slope:
  • It is composed of nutrient broth, glucose, and horse serum.
  • Used for cultivation of diphtheria, core bacterium
  • Dorset egg medium is used for the cultivation of diphtheria and tubercle bacilli.

Question 2. Mention three selective media with their uses
Answer:

Three selective media with their uses

Question 3. Robertson’s cooked meat media
Answer:

Robertson’s cooked meat media

It is an anaerobic culture media

Robertson’s cooked meat media Composition: Composed of Nutrient broth, Predigested cooked meat of ox heart

Robertson’s cooked meat media Uses:

• It is used for anaerobic culture
• For the preservation of the stock culture of the aerobic organism
• Valuable medium for preserving cultures of the delicate organism
• May be used as a recovery medium for spores

Culture Media And Culture Methods Short Answers

1. Blood agar
Answer:

Blood agar

• Blood agar is an enriched media
• In it blood is added as a nutritive substance to the basal media
• Though the blood agar is enriched media, the bacteria lysing red cells show a dearing around the colonies
• It helps in the growth of bacteria

Question 2. Blood culture
Answer:

Blood culture

Blood culture is one of the most important investigation in clinical microbiology

Blood culture Indications:

• Where there is a possibility of septicaemia or bacteriemia
• For diagnosis of pyrexia of unknown origin

Question 3. Transport media
Answer:

Transport media

For transport of specimens to foe laboratory, special media are devised and these are termed as transport media.

Examples:

1. Stuart’s transport medium:

• It is a non-nutrient soft agar gel containing a reducing agent to prevent oxidation and charcoal to neutralise bacterial inhibitors.
• It is used in organisms such as gonococci.

2. Buffered glycerol saline transport medium: Used for enteric bacilli.

Question 4. Enrichment media
Answer:

Enrichment media

• Some substances are incorporated in the liquid medium which have a stimulating effect on foe bacteria to be grown (or) inhibits its competitors.
• This results in an absolute increase in foe number of wanted bacteria related to other bacteria. Such media are called as enrichment media.
• They are very useful for the culture of faeces where foe non – pathogenic tend to overgrow than pathogenic ones.
• Example: Salmonella being overgrown by E. Coli.

Examples:

1. Tetrathionate broth:

• Composed of nutrient broth, Sodium thiosulphate, Calcium carbonate, and Iodine solution.
• Used for the culture of faeces, especially for salmonellae.

2. Selenite F broth

• Composed of Peptone water, Sodium selenite.
• For culture of Salmonellae, shigellae.

3. Alkaline peptone water: Peptone water at pH 9.0 was used for the culture of vibrio.

4. Robertson’s cooked meat broth:

• Composed of Nutrient broth, Predigested cooked meat of ox heart.
• Used for the culture of anaerobic bacteria.

Question 5. Preparation of blood agar media
Answer:

Preparation of blood agar media

• Blood agar is prepared by adding 10 ml of defibrinated sheep blood to 100 ml of nutrient agar
• The sterile nutrient agar is melted by steaming and then temperature is brought down to 45° C
• The required quantity of sheep blood is added aseptically with constant gentle shaking without froth formation
• The medium is immediately poured into the plates and allowed to set

Culture Media And Culture Methods Viva Voce

1. Robertson cooked meat media is an anaerobic media
2. Blood agar is an enriched media
3. Loeffler serum slope is composed of nutrient broth, glucose and horse serum

Growth And Nutrition Of Bacteria Important Notes

1. Bacterial growth curve

2. Bacterial nutrition

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Growth And Nutrition Of Bacteria Short Essays

Question 1. Growth curve
Answer:

Growth curve

The bacterial growth curve has four phases

Growth And Nutrition Of Bacteria Short Answers

Question 1. Classify bacteria based on growth temperature.
Answer:

Based on the growth temperature, bacteria are grouped into the following:

Growth And Nutrition Of Bacteria Viva Voce

1. Moisture is an essential ingredient of protoplasm
2. The thermal point is the lowest temperature that kills a bacterium under standard conditions in a given time
3. Lactobacillus is acidophilic bacteria
4. Vibrio cholera is a basophilic bacteria

Sterilization And Disinfection Important Notes

1. Disinfection:

• It is the destruction or removal of all pathogenic organisms or organisms capable of giving rise to infection

2. Sterilization controls:

Sterilization And Disinfection Long Essays

Question 1. Define sterilization. Describe an autoclave.
Answer:

Sterilization Definition:

• Sterilization is a process by which an article surface or medium is made free of all micro-organisms either in vegetative (or) spore form.

Autoclave:

• Autoclaving is the process of sterilization by saturated steam under high pressure above 100°C temperature.

Sterilization And Disinfection Questions And Answers

Autoclave Principle:

• Water boils when its vapour pressure equals that of the surrounding atmosphere
• When the atmospheric pressure is raised then the boiling temperature is also raised
• At normal pressure, water boils at 100° C but when the pressure inside a closed vessel increases, the temperature at which water boils also increases

Components of Autoclave:

• Autoclave is a modified pressure cooker (or) boiler.
• It consists of a vertical (or) horizontal cylinder made up of gunmetal (or) stainless steel in a supporting sheet iron case.
• The lid has screw clamps and is made airtight by an asbestos washer.
• Structures present in the lid are
• Discharge tap – for air and steam
• A pressure gauge
• Safety valve.
• Heating is generally done by electricity.

Autoclave Procedure:

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Sterilization Conditions:

Question 2. Write moist heat sterilization and dry heat method of sterilization.
Answer:

Moist Heat Sterilization:

• Moist heat kills micro-organisms by
  • Denaturation and coagulation of proteins.
• Methods of sterilization may be used at different temperatures as follows.

1. At a temperature below 100°C.

• Pasteurisation of milk: Two types of method
  • Holder method – 63°C for 30 min
  • Flash method – 72°C for 15 – 20 seconds
• Organisms like mycobacterium and brucellae are killed.
• Inspissation:
  • Inspissator is used.
  • The slow solidification of serum (or) egg is carried out at 80°C temperature for 30 minutes daily on 3 consecutive days.
• Vaccine bath:
  • Bacterial vaccines are sterilized in special vaccine baths at 60°C for one hour.

2. At a temperature 100°C

• Boiling:
  • Boiling for 10 to 30 min may kill most of vegetative forms but not spores.
  • Glass syringes, and rubber stoppers can be sterilized
• Tyndallisation:
  • Steam at 100°C for 20 minutes on 3 successive days is used.
  • Also known as intermittent sterilization.
  • The first exposure kills all vegetative forms
  • In the interval between the heating the remain¬ing spores germinate into vegetative forms, which are killed on subsequent heating.
  • Egg serum, sugar containing media can be steril¬ised.
• Steam steriliser at 100°C for 90 minutes.
  • Koch’s (or) Arnold’s steam steriliser is used.
  • Usually used for media which are decomposed at high temperature.

3. At a temperature above 100°C.

• Autoclave:
  • In this method material for sterilization is exposed to 121°C for 15 – 20 min at 15 lbs per square inch
  • Autoclave is used for culture media, rubber materials, syringes, and dressings

Dry Heat Sterilization:

The following procedures are under dry heat

Red heat

• Inoculating wires (or) loops, tips of forceps, and needles are held in the flame of a Bunsen burner till they become red hot

Flaming

• Glass slides, and scalpels are passed through bunsen flame without allowing them to become red hot

Incineration

• By this method infective material is reduced to ashes by burning.
• Solid dressings, animal carcasses, bedding, and pathological materials are dealt with in this method.

Hot air oven:

• Most widely used dry heat method of sterilization.
• Sterilization requires 160°C for 2 hours.
• Glasswares, surgical instruments, and chemicals can be sterilized.

Question 3. Classify sterilization. Write briefly about chemical methods of sterilization.
Answer:

• Sterilization Classification:
  • Physical methods:
    • Sunlight
    • Heat
      • Dry heat
      • Moist heat
    • Filtration
    • Radiation.
  • Chemical Methods:
    • Alcohols
    • Aldehydes
    • Phenols
    • Halogens
    • Oxidising agents
    • Salts
    • Surface active agents
    • Gases
    • Dyes

Chemical Methods of Sterilization:

Question 4. Write about physical methods of sterilization
Answer:

Sterilization And Disinfection Questions And Answers

Physical Methods of Sterilization:

Question 5. Define and differentiate sterilization and disinfection.
Answer:

Difference between sterilization and disinfection

Sterilization And Disinfection Short Essays

Question 1. Seitz filter
Answer:

Seitz filter

• Seitz filters are a type of asbestos filter.
• These are made up of asbestos (magnesium silicate)
• The filter disc is supported on a metal mount
• The filter is attached to a vacuum flask through a silicone rubber bung.
• After use the filter disc is discarded.
• These filters have a high absorbing capacity.

Seitz filter Disadvantage:

• The carcinogenic potential of the filters.

Seitz filter Uses:

• To sterilise sera, sugars, and antibiotic solutions
• Sterlisation of hydatid fluid.
• Purification of water.

Question 2. Hot air oven
Answer:

Hot air oven

• The most widely used sterilization method by dry beat

About oven:

• It is electrically heated and is fitted with a fan to ensure adequate and even distribution of hot air in the chamber.
• Thermostat is fitted to maintain the chamber air at a chosen temperature.

Hot air oven Temperature and Time:

• 160°C for 2 hours

Hot air oven Uses:

• Used for sterilization of
  • Glasswares like glass syringes, flasks, and test tubes
  • Surgical instruments like scalpels, and scissors.
  • Chemicals such as liquid paraffin, and fats.

Hot air oven Precautions:

• Should not be overloaded.
• Materials should be arranged in a manner that allows free circulation of air.
• Materials to be sterilized should be perfectly dry.
• Allow proper time for cooling at least 2 hours especially for glassware to prevent cracking.
• Any inflammable material should not be kept inside the oven.

Sterilisation Control:

• Spore test:
  • Non-toxigenic strains of cltetani are kept inside the oven.
  • Spores will be destroyed if sterilization is proper.
  • Browns tube with green – color spot is available. The green colour is produced after effective sterilization.

Question 3. Disinfection
(OR)
Antiseptics and disinfectants
Answer:

Antiseptics and disinfectants

• Antiseptic is an agent that destroys micro-organisms an contact and can be used on living tissue.
• A disinfectant is used on inanimate objects to de- struct all pathogenic organisms but not spores.

Disinfection Ideal Requirements:

• Have a wide spectrum of activity.
• Act in the presence of organic matter.
• Have high penetration power and quick action.
• Be safe and easy to use.
• Not cause local irritation.
• Be easily available and cheap.
• Not cause local irritation.
• Be easily available and cheap.
• Not corrode metals.
• Be stable
• Be effective in acidic as well as alkaline conditions

Disinfection Classification:

• Acids – Boric acid, benzoic acid.
• Alcohols – Ethanol, isopropyl alcohol.
• Aldehydes – Formaldehyde, glutraldehyde
• Surfactants – Soaps, cetrimide
• Cetyl pyridinium chloride.
• Phenol Derivatives; Phenol, cresol, Chlorhexidine, Hexachlorophene.
• Halogens: Iodine, Idophores, Chlorine, Chloramines.
• Oxidizing agents: Hydrogen .peroxide, Benzoyl peroxide
• Dyes: Gential violet, Methylene blue.
• Metallic salts: Silver nitrate, Zinc compounds.

Disinfection Uses in Dentistry:

• As a component of mouthwashes [chloroxylenol, chlor-hexidine]
• For gargling [potassium permagnates]
• For root canal therapy [ sodium hypochlorite]
• As gum paints [dequalinium chloride]

Question 4. Tyndallization
Answer:

Tyndallization

• Steam at 100°C for 20 minutes on 3 successive days is used.
• Also known as intermittent sterilization.
• The first exposure kills all vegetative forms
• In the interval between the heating the remaining spores germinate into vegetative forms, which are killed on subsequent heating.
• Egg, serum, and sugar-containing media can be sterilized.

Question 5. Cold sterilization
Answer:

Cold sterilization

• X-rays, gamma rays, and cosmic rays are ionizing radiations.
• This method is also known as cold sterilization.
• Have high penetration power and are highly lethal to DNA and other vital constituents
• They damage DNA by various mechanisms.
• Used for sterilization of disposable items such as plastic syringes, swabs, and culture plates.
• Gamma rays are commercially used for sterilization.

Sterilization And Disinfection Short Question And Answers

Question 1. Moist heat
Answer:

Moist heat

• Moist heat is one of the physical methods of sterilization

Moist heat Principle:

• Kills by denaturation and coagulation of proteins

Moist heat Types:

• This method may be used at different temperatures as follows:
• Temperature less than 100° C- pasteurization, in- aspirations, vaccine bath
• Temperature equal to 100° C- boiling, tyndillisation
• Temperature more than 100° C- autoclave

Question 2. Autoclave.
Answer:

Autoclave

• It is a method of moist heat sterilization
• It kills the micro-organisms by denaturation and coagulation of proteins
• In this method, material for sterilization is exposed to 121°C for 15-20 min at 15 lbs per square inch
• Uses: Used for sterilization of
  • Culture media
  • Rubber articles
  • Syringes and surgical instruments
  • OT gowns, dressing materials
  • Endodontic instruments
  • Hand instruments

Question 3. Pasteurization
Answer:

Pasteurization

• It is method of moist heat sterilization
• It kills the micro-organisms by denaturation and coagulation of proteins
• Temperature below 100°C is used
• Organisms like mycobacterium, and brucellae are killed by this process

Pasteurization Types:

• Holder method – 63°C for 30 min
• Flash method – 72°C for 15-20 seconds

Sterilization And Disinfection Viva Voce

1. A hot air oven is method of dry heat sterilization
2. Autoclave is a method of moist heat sterilization
3. Rideal Walker test is used to test the efficiency of a disinfectant
4. 2% glutaraldehyde is known as CIDEX
5. The germicidal effect of sunlight is due to its ultraviolet rays
6. Chlorhexidine is most effective against Gram-positive organism
7. Ionizing radiation are lethal to DNA