MCB 211: General Microbiology I

Part 2

Mrs Nasifat Adamu

1. MICROBIOLOGY METHODS AND INSTRUMENTATION

A. Microscopy:
It magnifies the sizes of microorganisms and aid in their identification.
Types of Microscopes:
 Electron Microscopy: uses electrons to create an image of the specimen.
 Light Microscopy: This include the followings:
 Bright Field Microscopy: It forms a dark image against a brighter background.
 Dark Field Microscopy: It forms a brighter image against a darker background.
 Phase-Contrast Microscopy.
 Fluorescence Microscopy.
Observation of Bacteria, Yeast and Moulds under Microscopic Magnification:
Yeast can be seen in unstained wet mounts at magnifications x100.
Bacteria are much smaller and can be seen unstained at x400.
A magnification of x1000 and the use of an oil immersion objective lens for observing stained
preparations are necessary for seeing bacteria characteristic shapes and arrangements.
Mould mycelium and spores can be observed in unstained wet mounts at magnifications of x100.

B. Wet Mount Slide Preparation:

This can be done with specific dyes or water. It is usually used to observe a little bit large
microorganisms or when studying microorganisms’ motility. A drop of the microorganisms can
be placed directly on the slide and observed. This is called wet mount.

C. Staining Techniques:

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It is necessary to stain microorganisms before they can be viewed with the light microscope.
Before staining, smear is usually prepared.

Preparation of Smear: A small sample of microorganisms is placed on a slide and air dry. The
smear is heat fixed by quickly passing it over a flame. This is called heat fixing. Heat fixing kills
the organisms, makes them adhere to the slide, and permits them to accept the stain.

There are two broad types of staining method: these are Simple and Differential Stain
Techniques.

i. Simple Stain Technique:

This involves the application of one stain to show cell shape and arrangement and, sometimes,
inclusions that do not stain, e.g. bacterial endospores. Simple stain techniques can either be one
of the followings:
 Positively Stain Technique: It is a Staining that can be performed with positively
charged basic dyes, such as crystal violet or methylene blue. They are attracted to
the negatively charged materials of the microbial cytoplasm.
 Negative Stain Technique: It is a Staining that can be performed with negatively
acidic charged dyes such as nigrosin or Congo red. They are repelled by the
negatively charged cytoplasm and gather around the cells, leaving the cells clear
and unstained.

ii. Differential Stain Technique:

This involves a sequence of several stains. It differentiates between different parts of a cell, e.g.
areas of fat storage or different groups of organisms, e.g. between Gram-positive and Gram-
negative bacteria. The followings are examples of differential staining techniques:

 Gram stain technique: Here bacteria are separated into two groups, Gram-
positive bacteria and Gram-negative bacteria.
Gram Staining Procedure: After smearing the test colony on slide, crystal violet is
first applied, followed by the mordant iodine, which fixes the stain. Then the
slide is washed with alcohol, and the Gram-positive bacteria retain the crystal-
violet iodine stain; however, the Gram-negative bacteria lose the stain. The Gram-

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 negative bacteria subsequently stain with the safranin dye, the counterstain.
Gram-negative bacteria appear red under the oil-immersion lens, while Gram-
positive bacteria appear blue or purple, reflecting the crystal violet retained during
the washing step.

The Gram stain procedure used for differentiating bacteria into two groups.

 Acid-fast technique: This technique differentiates species of Mycobacterium

from other bacteria.
D. Culture Techniques
Preparation of Culture Media:
 Powder media are re-hydrated according to manufacturer’s instructions.
Ingredients are completely dissolved, using heat if necessary.
 The dissolved media are dispensed into bottles or conical flask.

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  The media are then sterilization in the autoclave/pressure cooker as the case may
be.
Types of Media:
 Selective Media: It prevents the growth of certain bacteria while allowing others to
grow. E.g. Xylose Lysine Deoxycholate (XLD) agar, Brilliant Green Agar (BGA).
 Enrichment Media: It has special nutrients that enhance the growth of the desired
bacteria. E.g. Tryptic Soy Broth, Selenite-F Broth.
 Differential media: It contains a specific nutrient and an indicator that show whether the
species is able to use that nutrient. E.g. Lysine Iron Agar (LIA), Kligler Iron Agar (KIA).
Pouring a Plate
 It is a process of dispensing agar media into petri dishes.
Storage of Media
 Prepared media are stored at room temperature and away from direct sunlight.
 Cupboard and shelf are ideal place for storing media.
E. Inoculation Techniques:
It is the introduction of inoculum (microorganism) into culture media. The techniques include:
Streak Plate:
Wire loop is used for preparing a streak plate. This involves the progressive dilution of
inoculums of bacteria or yeast over the surface of solidified agar medium in a Petri dish in such
a way that colonies grow well separated from each other. The aim of the procedure is to obtain
single isolated pure colonies.

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 Streaked Plate.
Pour Plate
A pour plate is one in which a small amount of inoculum from broth culture is added by pipette
to a molten, cooled agar medium in a test tube or bottle, distributed evenly throughout the
medium, thoroughly mixed and then poured into a Petri dish to solidify. Pour plates allow
micro-organisms to grow both on the surface and within the medium. Most of the colonies
grow within the medium; few grow on the surface of the medium.
Spreading
Sterile spreaders are used to distribute inoculum over the surface of already prepared agar plates.
Wrapped glass spreaders may be sterilized in a hot air oven. They can also be sterilized by
flaming with alcohol.
F. Incubation
The lid and base of an agar plate should be taped together with 2-4 short strips of adhesive tape
as a protection from accidental (or unauthorized!) opening during incubation. (Although tape is
the preferred method, Parafilm could be used as an alternative for sealing the plates.) Agar plates
must be incubated with the medium-containing half (base) of the Petri dish uppermost otherwise
condensation will occur on the lid and drip onto the culture. This might cause colonies to spread
into each other and risk the spillage of the contaminated liquid.
G. Biochemical Tests
Indole Test:

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This test demonstrates the ability of certain bacteria to decompose the amino acid tryptophan to
indole.
Method: Inoculate 1% peptone water with one loop-full of culture and incubate at 37 0C for 24
hrs. Then add 0.5 ml Ehrlich’s reagent. A red colour indicates a positive reaction. E.g. E. Coli.
H2S Production Test:
The activity of some bacteria on sulfur containing amino acids frequently results in the liberation
of H2S. The H2S is usually tested for by demonstrating its ability to form black lead salt.
Method: Inoculate a loop-full of culture in 2% peptone water. Insert a lead acetate paper and
incubate at 37°C for 24 hrs. If H2S is produced, the blackening of lead acetate paper will take
place. E.g. Salmonella species.
Catalase Test:
This is a test performed to differentiate between Streptococcus and Staphylococcus.
Method: A portion of colony is picked up with a sterile wooden applicator stick and placed into
a drop of 3% Hydrogen peroxide (H2O2) on a clean slide. If bubbles develop within 10 seconds,
the organism is catalase positive and is it presumptively identified as Staphylococcus species.

Coagulase Test:
It is a test performed to differentiate o staphylococcus aureus and other staphylococci.
Method: A small amount of culture is mixed with coagulase plasma (rabbit plasma) on a slide or
in a tube. Presence of clumping on the slide shows positive result. The mixed culture and
coagulase plasma in tube is incubated for 4 hours and check at one hour intervals. The presence
of fibrin clot (thickening) indicate positive and is it presumptively identified as Staphylococcus
aureus.

H. DNA Methods

 PCR: Polymerase Chain Reaction, method used to amplify specific DNA sequences for
ease of identification and sequencing.
 Electrophoresis: DNA sample place in a gel substrate between two electrodes. The
charge and size of the DNA determines how fast the DNA migrates in the electrical field.
 DNA sequencing: Method to determine the order of which nucleotides are arranged on a
strand of DNA.

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 I. Aseptic Techniques: Method to collect, grow and preserve a sample such that no
other microbes are introduced or lost from the original culture.
Malam Tasiu

2. STERILIZATION AND DISINFECTION:

Sterilization:
It means the complete destruction of all the micro-organisms including spores from an object or
environment. It is usually achieved by heat, filtration, chemicals or radiation.
 Use of Autoclave: Steam under pressure is used to produce a temperature of 121ºC and
held for 15 minutes; all micro-organisms including bacterial endospores will be
destroyed. Media, solutions and glassware are sterilized using autoclave.
 Flaming: Wire loops, glass spreader, bottles’ necks and test tubes’ necks are sterilized
using red heat in a Bunsen flame. The wire loop is sterilized before and after use. The
loop must be heated to red hot to make sure that any contaminating bacterial spores are
destroyed.
 Hot Air Oven: Empty glassware, glass pipettes and petri dishes (glasses) are sterilized
using hot air oven. The material are wrapped in either grease proof paper or aluminum
and held at 160ºC for 2 hours.
 Ultra-violet Radiation: Media rooms contain ultra-violet fluorescent which is used to
sterilized media plates.
 Alcohol: Glass spreader can be sterilized using small volume of 70% alcohol (70%
industrial methylated spirit) contained in a vessel with a lid (either a screw cap or
aluminium foil). The vessel is passed quickly through a Bunsen burner flame to ignite the
alcohol; the alcohol will burn and sterilize the glass. The spreader is removed from the
flame and the alcohol is allowed to burn off. Sterilized spreader is not kept on the bench.

Disinfection:
Is the inhibition or removal of microbes that may cause disease or other problems e.g.
contamination or spoilage. It is usually achieved by the use of chemicals. The chemical used
are usually referred to as disinfectants.

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  Hypochlorite (sodium chlorate I): It is used in discard pots for pipettes and slides.
 Ethanol (70% industrial methylated spirit). It is used in disinfecting work benches.

3. INDUSTRIAL USES OF MICROORGANISMS

Large cultures of bacteria and other microorganisms are used to produce various industrial
products. At first, selection and mutation were the major means of improving microbial cultures
for production of industrial products. With advent of biotechnology, recombinant DNA
techniques are now employed to manipulate genetic information and design desire products. The
industrial products produced by microorganisms are: Amino acids, organic acids, enzymes,
pharmaceuticals, medical compounds, food supplements, fuel and energy-related products.
Microbial Industrial Products:
i. Primary Metabolites:
These consist of compound related to synthesis of microbial cells and occur in the growth phase
(trophophase). This include: Amino acids, fermentation end products (such as ethanol and
organic acids) and industrial enzymes.
Amino Acids: These are produced by Corynebacterium dismutans, Corynebacterium
glutamicum and Escherichia coli. They are used in the food industry as nutritional supplements
in bread products, e.g. lysine and glutamic acid. They can also be used as flavor-enhancing
compound, e.g. monosodium glutamate (MSG).
Organic Acids: These are produced by Aspergillus niger, Acetobacter and Rhizopus nigricans.
These acids are citric, acetic, lactic, fumaric and gluconic acids. Citric acid is used in food,
beverage and pharmaceutical industries. Gluconic acid can be used as carrier for calcium and
iron and as a component of detergents.
Enzymes: e.g.
Amylase: It is produced by Bacillus subtilis. It is use in the production of textiles, food and
drinks.
Proteases: It is produced by Bacillus spp. It is used for clarifying beer, tenderizing meat. It is
also used in toothpaste to reduce plaque. It is also used in laundry detergents to help clean.
ii. Secondary Metabolites:

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They usually accumulate during the period that follows the active growth phase, called
idiophase. They have no direct relationship to the synthesis of cell materials and normal growth.
Most antibiotics and Mycotoxins are secondary metabolites products. e.g.
Penicillin: It is produced by Penicillium chrysogenum. It is used in treating infectious disease
caused by gram-positive bacteria.
Streptomycin: It is produced by Streptomyces griseus. It is used in treating infectious disease
caused by gram-negative bacteria and Mycobacterium toberculosis.
Nystatin: It is produced by Streptomyces noursei. It is used in treating fungal disease.
Cephalosporin: It is produced by Cephalosporium acremonium. It is a broad spectrum
antibacterial.
Production of Desire Products using Recombinant DNA:
Genes for human proteins are inserted into bacteria and cultures of these bacteria now produce
commercial quantities of: human insulin, human growth hormone, human somatostatin and
human interferon.

4. MICROORGANISMS IN FOODS:
Microorganisms can be used to transform raw foods into gastronomic delights such as cheeses,
yogurt, pickles, and beverages (Coffee, Cocoa). Wines, beers and other alcoholic products can
also be produced through microbial activity. On the other hand, the growth of certain microbes in
food may be detriment, because the microbes may be food poisoning organisms, Infectious
microorganisms or food spoilage microorganisms, there by affecting human health and food
quality.
Food Fermenting Microorganisms:
Fermenting bacteria are used to make a wide variety of foods such as bread, bakery products,
yogurt, cheeses, etc. Many of the smells and flavors of these food products are due to activity of
the bacterial used. Culture of different bacterial species; often with several fungal species are
used to produce different kinds of fermented foods. Some of the examples of foods produce
through the activities of microbes are:
Cheeses: It is often produced by the actions of groups of microorganisms including bacteria and
fungi. The first step in making most cheeses is to prepare a curd by adding lactic acid bacteria
(Lactococcus lactis, or Lactobacillus bulgaricus) and rennin or bacterial enzymes to milk. The

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bacteria sour the milk and enzymes coagulate the milk, protein and casein, to produce a soft
‘curd’ to make cheese and liquid ‘whey’ which is a waste product. The amount of whey
removed determines the hardness of the cheese. eg. Soft cheeses, the whey are simply drained
away. Harder cheeses are heated and pressurized to remove additional whey from the mixture.
After this separation, most cheeses are ripened with inoculations of various species of bacteria
and/or fungi, e.g. Leuconostoc cremoris, Penicillium camemberti or Brevibacterium linens.
Yogurt: It is produced by a special starter culture in which two major bacteria are present in a
1:1 ration: Sreptococcus thermophilus and Lactobacillus bulgaricus. Acid in the yogurt is
produced by Sreptococcus thermophilus, and aroma components are formed by Lactobacillus
bulgaricus. Freshly prepared yogurt contains 109 bacteria per gram.
Pickles: Pickles are made by fermenting cucumbers in a mixture of bacteria normally found
growing on them. e.g. leuconostoc mesenteroids, Enterococcus faecalis, Pediococcus cerevisiae,
Lactobacillus brevis and Lactobacillus plantarum.
Bread: In bread making, yeast growth is carried out under aerobic conditions. The addition of
yeast causes the bread to rise. Light texture of the bread is as a result of production of CO 2 by the
yeast.
Food Poisoning Microorganisms:
These microbes are commonly found on many raw foods. Poor personal hygiene, improper
cleaning of storage and preparation areas and unclean utensils cause contamination of raw and
cooked foods. There are two types of food poisoning organisms. These are:
a. Those which cause decease by infection: They must grow in food in large numbers and
cause infection when consumed together with food. The most common microorganisms in this
category include Salmonella, enteropathogenic E. coli, Vibrio parahaemolyticus, Yersinia
enterocolytica etc.
e.g. Salmonella Typhimurium: The gastrointestinal tracts of animals and man are common
sources of Salmonella. High protein foods such as meat, poultry, fish and eggs are most
commonly associated with Salmonella. The major causes of salmonellosis are contamination of
cooked foods and insufficient cooking. Contamination of cooked foods occurs from contact with
surfaces or utensils that were not properly washed after use with raw products.
b. Those which produce toxin in food: They cause intoxication and must grow in food in
large numbers and produce enough toxin and when consumed together with food, they cause

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intoxication. The most common microorganisms in this category includes, Clostridium
botulinum, Staphylococcus aureus and toxigenic fungi e.g. Aspergillus flavus.
e.g. Staphylococcus aureus: When S. aureus is allowed to grow in foods, it can produce a toxin
that causes illness. Although cooking destroys the bacteria, the toxin produced is heat stable and
may not be destroyed. Staphylococcal food poisoning occurs most often in foods that require
hand preparation, such as potato salad, and sandwich spreads.
e.g. Clostridium botulinum: It causes Botulism. It occurs mostly in home-canned foods. Cl.
botulinum can exist as a heat-resistant spore, and can grow and produce a neurotoxin in under
processed, home-canned foods. An affected food may show signs of spoilage such as a bulging
can or an off-odor. The botulinum toxin is destroyed by boiling the food for 10 minutes.
Infectious Microorganisms:
These are organisms whose presence in small numbers in food and when consumed can cause
infection. In this case, the food acts as a vector but not necessarily as a growth medium.
Infectious organisms can be transmitted by various ways including man to man and are said to be
contagious. Organisms in this group includes; Vibrio cholerae O1, Salmonella Typhi, Shigella
sonnei, Bacillus anthracis, Hepatitis B virus etc.
Spoilage Organisms:
Spoilage organisms are the organisms whose growth in the food creates undesirable
characteristics in that food such as loss of fruit structure, food bitterness, souring, bad odor,
acidification, rancidity and putrefaction. Any microorganism which is not intentionally added
into food or intentionally allowed to grow in food so as to impart certain qualities in that food is
considered a contaminant. Growth of the contaminant in that food will spoil the food making it
unfit for human consumption. Some useful microorganisms, e.g. lactic acid bacteria are
considered as spoilage organisms in fruit juices but not in milk.

5. PATHOGENIC ROLE OF MICROORGANISMS:

Pathogen: It is a microorganism (or virus) that is able to produce disease.
Pathogenicity: It is the ability of a microorganism to cause disease in another organism. The
ability of microorganism to harm its host depends on its ability to proliferate in the host body and
to produce toxins which damage or kill the host cells.

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Potential Pathogens: They are normal bacterial floras (e.g. Staphylococcus aureus,
Streptococcus pneumoniae) that live in a commensal relationship without producing disease
unless they have an opportunity brought on by some compromise or weakness in the host's
anatomical barriers, tissue resistance or immunity.
Opportunistic Pathogens: These are bacteria that cause diseases in immune compromised host
which typically would not occur in a healthy (non-compromised) host. A member of the normal
flora such as Staphylococcus aureus or E. coli can cause an opportunistic infection disease, but
so can an environmental organism such as Pseudomonas aeruginosa. Pseudomonas aeruginosa
is one of the most common opportunistic pathogens of humans. The bacterium causes urinary
tract infections, respiratory system infections, dermatitis, soft tissue infections, bacteremia and a
variety of systemic infections, particularly in cancer and AIDS patients who are
immunosuppressed.
Obligate Pathogens: These are pathogens that do not associate with their host except in the case
of diseases. e.g. Mycobacterium tuberculosis, Bacillus anthracis, Neisseria gonorrhea etc.
Determinants of Virulence (Virulence Factors): Pathogenic microorganisms are able to
produce disease because they possess certain structural or biochemical or genetic traits that
render them pathogenic or virulent. These traits are referred to as virulence factors. Some
pathogens may rely on a single determinant of virulence (virulence factor), such as toxin
production, to cause damage to their host. Examples:
Clostridium tetani and Corynebacterium diphtheria are able to produce disease because they
possess the ability to produce a toxin.
Pathogenic Salmonella possess a lipopolysaccharide which inhibits the C5b–9 complement
complex from attacking the hydrophobic domains of the bacterial outer membrane.
Pathogenic Neisseria, Haemophilus influenzae type b (Hib), and Streptococcus pneumonia
possess antiphagocytic polysaccharide capsules which they use in avoiding phagocytosis
Other pathogens, such as Staphylococcus aureus, Streptococcus pyogenes and Pseudomonas
aeruginosa, maintain a large repertoire of virulence determinants and consequently are able to
produce a more complete range of diseases that affect different tissues in their host.
Examples of diseases in human cause by certain bacteria are as follows:
Tuberculosis: It is caused by Mycobacterium tuberculosis. It is a Respiratory disease. Infection
usually occurs in lungs and cause formation of ‘tubercles’. It is most commonly acquired by

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inhaling. Ninety percent of infected people remain infected for life but never develop symptoms
of the disease (i.e. asymptomatic). A good example of a balanced host/parasite relationship.
Hosts are usually not aware of pathogen and it is usually eliminated by the immune system. If
body’s defenses fail, disease results.
Symptoms: Weight loss, coughing bloody cough, fatigue, weakness, anorexia, low grade fever.
Anthrax: It is caused by Bacillus anthracis. It is a common soil microorganism. It grows slowly in soil
and produces spores that can persist for years up to 60 years. It is a zoonosis disease (animal disease) that
people can catch. It is a fatal disease of livestock; eg. Sheep, cattle, goats, horses. People catch it from
animals, not usually from other people. Spores can persist in humans. It is mainly an occupational
disease: farmers, vets, textile and fur workers are more liable. It enters through cuts and abrasions on skin
or spores can be inhaled.
Types of Anthrax:
a. Cutaneous Anthrax: If spores enter skin through abrasions or cuts, it begins as a skin
infection of pustules and develops into a necrotic ulcer. Bacilli release toxins that cause
local swelling. If untreated may spread through lymphatic system to blood to cause
septicemia. If so, it will cause shock and death within a few days.
b. Pulmonary anthrax: It is the most dangerous form; with higher mortality rate. It can be
fatal within hours. Spores are inhaled. High exposure to spores is required to get the
disease.
Gonorrhea: It is caused by Neisseria gonorrhea. It is a sexually transmitted disease (STD).
Symptoms: Pain and discharge at the infected site. In men, this is typically the urethra, and in
women, the uterine cervix. Fever and lower abdominal pain, a syndrome called pelvic
inflammatory disease (PID). In women, sterility or ectopic pregnancy may occur.
Examples of diseases in human cause by certain virus are as follows:
Acute Respiratory Disease (ARD): The viruses that cause ARD include influenza viruses,
parainfluenza viruses, rhinoviruses, adenoviruses, respiratory syncytial virus (RSV), and
respiratory coronaviruses. This disease include:
a. Influenza: This is caused by Influenza virus types A, B and C. Influenza virus
type A and B typically cause more severe symptoms than influenza virus type C.
Symptoms: Fever, diffuse muscle aches and chills. This is followed within 12 to
36 hours by respiratory signs, such as rhinitis, cough, and respiratory distress. The
acute phase usually lasts 3 to 5 days, but a complete return to normal activities

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 may take 2 to 6 weeks. Serious complications, especially pneumonia, are
common.
b. Respiratory Syncytial Virus Disease: It is caused by respiratory syncytial virus
(RSV). RSV primarily infects the bronchi, bronchioles, and alveoli of the lung.
The illnesses clinically categorized as croup, bronchitis, bronchiolitis or
pneumonia and are extremely common in infants. The acute phase of cough,
wheezing and respiratory distress lasts 1 to 3 weeks. Elderly or
immunocompromised patients are also frequently susceptible and can be severely
affected.
c. Parainfluenza Disease: It is caused by Parainfluenza 1, 2, 3 and 4 viruses. They
cause serious diseases they can cause in infants and young children. The onset of
the illness may be abrupt, as in acute sporadic croup, but usually begins as a mild
upper respiratory infection (URI) with variable progression over 1 to 3 days to
involvement of the middle or lower respiratory tract. Duration of acute illness can
vary from 4 to 21 days but is usually 7 to 10 days.
 Measles: It is caused by measles virus which is classified in the paramyxovirus family
and genus Morbillivirus. Measles infections often produce severe illness in children,
associated with high fever, widespread rash, and transient immunosuppression.
 Rubella (German measles or 3-day measles): It is caused by rubella virus which is
classified as a member of the togavirus family. Infections by rubella virus are often mild,
or even asymptomatic. The major concerns are the profound effects on developing
fetuses, resulting in multiple congenital malformations.
Examples of diseases in human cause by certain fungi are as follows:
 Dermatophytoses: They are superficial infections of the skin and its appendages. They
commonly known as ringworm, athlete’s foot, and jock itch. They are caused by species
of the genera Microsporum, Trichophyton, and Epidermophyton, which are collectively
known as dermatophytes.
 Candidiasis: It is caused by Candida albicans. It occurs in localized and disseminated
forms. Localized disease is seen as erythema and white plaques in moist skin folds
(diaper rash) or on mucosal surfaces (oral thrush). It may also cause the itching and thick
white discharge of vulvovaginitis. Deep tissue and disseminated disease are limited

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 almost exclusively to the immunocompromised. Diffuse pneumonia and urinary tract
involvement are especially common.
 Aspergillosis: This can be caused by Aspergillus fumigates, Aspergillus flavus and
Aspergillus niger. Aspergillus can cause clinical allergies or occasional invasive
infection, leading to allergic and invasive aspergillosis respectively. In both cases, the
lung is the organ primarily involved.

6. IDENTIFICATION AND ECONOMIC IMPORTANCE OF SOME SELECTED

MICROBIAL GROUPS:

a) Genus Neisseria:
Belong to the family Neisseriaceae. The species that are of economic important are Neisseria
gonorrhoeae (gonococci) and Neisseria meningitidis (meningococci). They are pathogenic
for humans and typically are found associated with or inside polymorphonuclear cells.

Morphology of Neisseria:

The typical neisseria is a gram-negative, nonmotile diplococcus, approximately 0.8 m in

diameter. Individual cocci are kidney-shaped. When the organisms occur in pairs, the flat or
concave sides are adjacent. On enriched media (eg, Mueller-Hinton, modified Thayer-Martin),
gonococci and meningococci form convex, glistening, elevated, mucoid colonies of 1–5 mm in
diameter. Colonies are transparent or opaque, nonpigmented, and nonhemolytic.

Laboratory Identification of Neisseria:

Specimens: Pus and secretions are taken from the urethra, cervix, rectum, conjunctiva, throat, or
synovial fluid for culture and smear.

Stained Smears: Gram-stained smears of urethral or endocervical exudates reveal many gram-
negative diplococci within pus cells. These give a presumptive diagnosis.

Culture: Pus or mucus is streaked on enriched selective medium (eg, modified Thayer-Martin
medium) and incubated in an atmosphere containing 5% CO 2 (candle extinction jar) at 37 °C. To
avoid overgrowth by contaminants, the selective medium contains antimicrobial drugs (eg,

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vancomycin, 3 g/mL; colistin, 7.5 g/mL; amphotericin B, 1 g/mL; and trimethoprim, 3 g/mL).
Meningococci and gonococci grow best on media containing complex organic substances such as
heated blood, hemin, and animal proteins.

Oxidase Test: The neisseriae produce oxidase and give positive oxidase reactions. The oxidase
test is a key test for identifying them. When bacteria are spotted on a filter paper soaked with
tetramethylparaphenylenediamine hydrochloride (oxidase), the neisseriae rapidly turn dark
purple.

Economic Importance of Neisseria:

Neisseria gonorrhoeae (Gonococci): In males, there is usually urethritis, with yellow, creamy
pus and painful urination. Fibrosis may occur, sometimes leading to urethral strictures.

In females, there is salpingitis, fibrosis, and obliteration of the tubes. Infertility occurs in 20%
of women with gonococcal salpingitis.

Neisseria meningitidis (meningococci): When the organisms reach the bloodstream via
nasopharynx, bacteremia occurs. There is also Fulminant meningococcemia which is more
severe, with high fever and hemorrhagic rash. Meningitis is the most common complication of
meningococcemia. It usually begins suddenly, with intense headache, vomiting, and stiff neck,
and progresses to coma within a few hours.

b) Genus Staphylococcus:

The genus Staphylococcus has at least 35 species. The three main species of clinical importance
are Staphylococcus aureus, Staphylococcus epidermidis, and Staphylococcus saprophyticus.
Staphylococcus aureus is coagulase-positive, which differentiates it from the other species.

Morphology of Staphylococcus

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Staphylococci are spherical cells about 1m in diameter arranged in irregular clusters. Single
cocci, pairs, tetrads, and chains are also seen in liquid cultures. Young cocci stain strongly gram-
positive; on aging, many cells become gram-negative. Staphylococci are nonmotile and do not
form spores. Colonies on solid media are round, smooth, raised, and glistening. S aureus usually
forms gray to deep golden yellow colonies. S epidermidis colonies usually are gray to white.

Laboratory Identification of Staphylococcus:

Specimens: Surface swab pus, blood, tracheal aspirate, or spinal fluid.

Smears: Staphylococci appear as gram positive cocci in clusters in Gram-stained smears of pus
or sputum.

Culture: Staphylococci grow under aerobic or microaerophilic conditions. They grow most
rapidly at 37 °C but form pigment best at room temperature (20–25 °C). Many colonies develop
pigment upon prolonged incubation. No pigment is produced anaerobically or in broth. Various
degrees of hemolysis are produced by S aureus and occasionally by other species. Specimens
planted on blood agar plates give rise to typical colonies in 18 hours at 37 °C, but hemolysis and
pigment production may not occur until several days later and are optimal at room temperature.
Specimens contaminated with a mixed flora can be cultured on media containing 7.5% NaCl; the
salt inhibits most other normal flora but not S aureus. Mannitol salt agar or commercially
available chromogenic media are used to screen for nasal carriers of S aureus and patients with
cystic fibrosis.

Catalase Test: This test is used to detect the presence of cytochrome oxidase enzymes. A drop
of 3% hydrogen peroxide solution is placed on a slide, and a small amount of the bacterial
growth is placed in the solution. The formation of bubbles (the release of oxygen) indicates a
positive test. The catalase test differentiates the staphylococci, which are positive, from the
streptococci, which are negative.

Coagulase Test: Citrated rabbit (or human) plasma diluted 1:5 is mixed with an equal volume of
broth culture or growth from colonies on agar and incubated at 37 °C. A tube of plasma mixed
with sterile broth is included as a control. If clots form in 1–4 hours, the test is positive.

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Coagulase-positive staphylococci are considered pathogenic for humans; however, coagulase-
positive staphylococci of dogs (Staphylococcus intermedius) and dolphins (Staphylococcus
delphini) rarely cause disease in humans. Infections of prosthetic devices can be caused by
organisms of the coagulase-negative S epidermidis group.

Economic Importance of Staphylococcus

S aureus is a major pathogen for humans. Almost every person will have some type of S aureus
infection during a lifetime, ranging in severity from food poisoning or minor skin infections to
severe life-threatening infections. The coagulase-negative staphylococci are normal human flora
and sometimes cause infection, often associated with implanted appliances and devices,
especially in very young, old, and immunocompromised patients. Approximately 75% of these
infections caused by coagulase-negative staphylococci are due to S epidermidis; infections due
to Staphylococcus lugdunensis, Staphylococcus warneri, Staphylococcus hominis, and other
species are less common. S saprophyticus is a relatively common cause of urinary tract
infections in young women. Other species are important in veterinary medicine.

c) Enterobacteriaceae:

The Enterobacteriaceae are a large, heterogeneous group of gram-negative rods whose natural
habitat is the intestinal tract of humans and animals. The family includes many genera
(Escherichia, Shigella, Salmonella, Enterobacter, Klebsiella, Serratia, Proteus, and others).
Some enteric organisms, eg, Escherichia coli, are part of the normal flora and incidentally cause
disease, while others, the salmonellae and shigellae, are regularly pathogenic for humans. The
Enterobacteriaceae are facultative anaerobes or aerobes, ferment a wide range of carbohydrates,
possess a complex antigenic structure, and produce a variety of toxins and other virulence
factors. Enterobacteriaceae may be called enteric bacteria or coliforms.

Morphology of Shigellae:

Shigellae are slender gram-negative rods; coccobacillary forms occur in young cultures.
Shigellae are facultative anaerobes but grow best aerobically. Convex, circular, transparent
colonies with intact edges reach a diameter of about 2 mm in 24 hours.

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Laboratory Identification of Shigellae:

Specimens: Specimens include fresh stool and rectal swabs for culture.

Smear: Slender gram-negative rods. Large numbers of fecal leukocytes and some red blood cells
often are seen microscopically.

Culture: The specimens are streaked on differential media (eg, MacConkey's or EMB agar) and
on selective media (Hektoen enteric agar or salmonella-shigella agar). Colorless colonies are
seen and inoculated into triple sugar iron agar. Organisms that fail to produce H 2S, that produce
acid but not gas in the butt and an alkaline slant in triple sugar iron agar medium, and that are
nonmotile are suspected to be shigella.

Economic Importance of Shigellae:

Shigellae infectious disease is accompany by abdominal pain, fever, and watery diarrhea.
However, in children and the elderly, loss of water and electrolytes may lead to dehydration,
acidosis, and even death. The illness due to S dysenteriae may be particularly severe.

Morphology of Salmonella: Gram-negative rods. Most isolates are motile with peritrichous
flagella. Salmonella possess circular, convex, smooth colonies with distinct edges.

Laboratory Identification of Salmonella:

Specimens: Blood, stool, bone marrow and urine.

Culture: Specimen streaked on xylose lysine deoxycholate (XLD) and brilliant green agar
(BGA) agar and incubated overnigth, the colonies will be red with black centre and red only
respectively. Suspect colonies from solid media will be identified by biochemical test and slide
agglutination tests with specific sera.

Agglutination Test: In this test, known sera and suspected culture are mixed on a slide.
Clumping, when it occurs, can be observed within a few minutes. This test is particularly useful

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for rapid preliminary identification of cultures. There are commercial kits available to
agglutinate and serotype Salmonellae into S. Typhi, S. Paratyphi A, B and C and S. Enteritidis.

Economic Importance of Salmonellae:

Salmonella Typhi, Salmonella Choleraesuis, and perhaps Salmonella Paratyphi A and

Salmonella Paratyphi B are primarily infective for humans, and infection with these organisms
implies acquisition from a human source. The vast majority of Salmonellae, however, are chiefly
pathogenic in animals that constitute the reservoir for human infection: poultry, pigs, rodents,
cattle, pets (from turtles to parrots), and many others.

The organisms almost always enter via the oral route, usually with contaminated food or drink.
The mean infective dose to produce clinical or subclinical infection in humans is 10 5–108
Salmonellae (but perhaps as few as 103 Salmonella Typhi organisms). Salmonellae produce three
main types of disease in humans, but mixed forms are frequent. These are: Enteric fevers
(Typhoid fever), bacteremia with focal lesions and Enterocolitis.

7. MICROBIAL VARIATION AND HEREDITY:

Heredity:

It is the movement of information from parent to offspring. The information is carried by nucleic
acids. The nucleic acid is DNA. For some (but certainly not all) viruses, that nucleic acid is
RNA.

Gene: It is a specific segment of DNA that is the basic unit of heredity. It is a linear sequence of
nucleotides that form a functional unit of chromosome. When any living organism reproduces, it
passes on genetic information to its offspring. This information takes the form of genes. The total
complement of an organism’s genetic material is called its genome.

Chromosome: The polymer of nucleic acid (i.e., long chain of linked together nucleotides) is
called chromosome. In cellular forms of life that polymer is DNA and a single chromosome
consists of that DNA hydrogen bonded with a complementary strand of DNA, i.e., as a double

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helix. The bacterial chromosome is a long circle of deoxyribonucleic acid (DNA) that is
attached to the membrane of the cell. During replication, the chromosome is copied, and the two
copies are divided into the two daughter cells, there by transferring genetic information into the
two daughter cells. Transfer of genetic information from the mother cell to offspring is called
vertical transmission.

Plasmid (Extrachromosomal DNA): A plasmid is a small circle of DNA carrying only a few
genes; it is replicated independently of the "main" chromosome. Some plasmids allow bacteria to
engage in bacterial conjugation in which a pilus joins two bacteria to permit the transfer of
plasmid DNA.

Transposon: It is a moveable genetic element that cannot self-replicate. Transposons contain

insertion sequences and can transfer their information by inserting themselves into other loci in
the same or other genetic elements (e.g., plasmids, chromosomes, viral DNA). They carry
specialized information.

Bacteria Variation:

Any change in the genotype of a bacterium or its phenotype is known as variation.

Genotypic variation: It can occur as a result of changes in the genes by way of mutation, loss or
acquisition of new genetic elements. These variations are heritable.

Phenotypic variations: They are seen temporarily when bacteria are grown under certain
environmental conditions. These variations are not heritable.

Heritable variations: These are caused by the followings:

Mutation: A gene will mutate spontaneously, about once in a hundred million cell divisions.
Such bacteria are called mutants. Most of these mutants die, but when a mutant can adapt itself
to the environment more readily; it may emerge as a new variant. Chromosomal mutations may
lead to Emergence of drug resistance in bacteria. Examples include methicillin resistance in
Staphylococcus aureus, Multi-drug resistance in Mycobacterium tuberculosis.

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Transformation: Some bacteria have ability to uptake naked DNA fragment from the
surrounding environment. When such a DNA aconfers new property to the bacterium, it is
termed transformation. Change from R form of Streptococcus pneumoniae to S form is due to
transformation.

Conjugation: Transfer of genetic material (usually plasmids) from one bacterium to another
through the mediation of sex pili is known as conjugation. Any property that is coded on a
transmissible plasmid can be transferred to a recipient bacterium. Properties such as drug
resistance mediated by beta-lactamases, bacteriocin production etc can be transferred by
conjugation.

Transduction: Transfer of genetic material through mediation of bacteriophage is known as

transduction. Only those strains of Corynebacterium diphtheriae that are infected by a beta
phage are toxigenic. Change in O antigen in Salmonella (S. Anatum->S. Newington->
S.Minneapolis) is because of lysogenic phage.

Transposition: Variations in the flagellar antigens in Salmonella are due to transposons. Similar
gene rearrangements may result in antigenic variations, as in Neisseria gonorrhoeae and
Borrelia recurrentis.

Non-heritable variations:

A variation in the phenotype of a microorganism, where the genetic constitution remains

unchanged is a non-heritable variation. Such variations are seen due to a change in
environmental conditions and such variations are neither permanent nor heritable. They may
revert back to normal state when the conditions are restored.

Some examples are:

 Loss of flagella in Salmonella typhi when grown in phenol agar (H-O variation)
 Pleomorphism (variation in shape) in old cultures
 Lack of pigment production by S.aureus in anaerobic conditions
 Formation of spheroplasts and protoplasts

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  V-W variation in Salmonella typhi that is characterized by loss of Vi antigen
 S-R variation in Salmonella typhi that is characterized by loss of O antigen and change in
colony morphology to rough type.
 Production of flagella in Listeria monocytogenes occurs at temperature less than 20oC.

REFERENCE

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