Bacteriology

Basics
Morphology, Classification, Staining Methods

Dr.T.V.Rao MD

Introduction:
Microorganisms several classes of living beings Based on the organization of their cellular structures, all living cells can be divided into two groups: eukaryotic and prokaryotic
Eukaryotic cell types - Animals, plants, fungi, protozoans, and algae Prokaryotic cell types - bacteria & blue green algae
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Antioni van Leeuwenhoek

Leeuwenhoek is called "the inventor of the microscope"
Created a simple microscope that could magnify to about 275x, and published drawings of microorganisms in 1683 Could reach magnifications of over 200x with simple ground lenses
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How a Microscope Works with..

Ocular Lens (Magnifies Image) Objective Lens (Gathers Light, Magnifies And Focuses Image Inside Body Tube)
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Body Tube (Image Focuses)

Bending Light: The objective (bottom) convex lens magnifies and focuses (bends) the image inside the body tube and the ocular convex (top) lens of a microscope magnifies it (again).

The Light Microscope

many types
bright-field microscope dark-field microscope phase-contrast microscope fluorescence microscopes
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compound microscopes
image formed by action of 2 lenses
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The Compound Microscope

The Optical System
Objective Lens: the lens closest to the specimen; usually several objectives are mounted on a revolving nosepiece.
Parafocal: when the microscope is focused with one objective in place, another objective can be rotated into place and the specimen remains very nearly in correct focus.
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Eyepiece or Ocular Lens: the lens closest to the eye.

Monocular: a microscope having only one eyepiece 6 Binocular: a microscope having two eyepieces.

Phase Contrast Microscopy

light rays through objects of different change in phase, not intensity special ring-shaped condenser diaphragm special glass disc in objective
change phase differences to intensity differences can view transparent objects as dark on light background (without staining)

Right; human brain glial cells

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The Bright-Field Microscope

Produces a dark image against a brighter background Has several objective lenses
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par focal microscopes remain in focus when objectives are changed

total magnification
product of the magnifications of the ocular lens and the objective lens
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Fluorescence Microscopy
Illuminate specimen with UV visible fluorescence (filter removes harmful UV) View auto-fluorescent objects (e.g., chloroplasts) Stain with specific fluorescent dyes, which absorb in region 230-350 nm & emit orange, yellow or greenish light Images appear coloured against a dark background

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Schematic of typical animal (eukaryotic) cell, showing subcellular components. Organelles: (1) nucleolus (2) nucleus (3) ribosome (4) vesicle (5) rough endoplasmic reticulum (ER) (6) Golgi apparatus (7) Cytoskeleton (8) smooth ER (9) mitochondria (10) vacuole (11) cytoplasm (12) lysosome (13) centrioles
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Background Information
Prokaryotes

Prokaryotes represent two domains, bacteria and archaea. Archaea live in Earths extreme environments. Bacteria are the most abundant and diversified organisms on Earth.
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Eukaryotes have organelles

Much larger; more complex than prokaryotes Processes compartmentalized into organelles
Nucleus Protein synthesis (ribosomes, RER, Golgi) Mitochondria; chloroplasts Lysosomes Plasma membranes have different modifications Cytoskeleton

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Differences between prokaryotic & eukaryotic cells

Character Nucleus Nuclear membrane Prokaryotes Absent Eukaryotes Present

Nucleolus

Absent

Present
One or more paired and linear Mitosis fluid phospholipid bilayer containing sterols Capable of endocytosis and exocytosis
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Chromosome One circular Cell division Binary fission

Cytoplasmic Structure and fluid phospholipid membrane Composition bilayer, lacks sterols Function Incapable of endocytosis (phagocytosis and pinocytosis) and exocytosis Dr.T.V.Rao MD

Differences between prokaryotic & eukaryotic cells

Character Cytoplasm Mitochondria Prokaryotes Absent Eukaryotes Present

Lysosomes Golgi apparatus

Absent Absent

Present Present

Endoplasmic reticulum Vacuoles Ribosomes

Absent

Present

Absent 70 S
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Present 80 S
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Differences between prokaryotic & eukaryotic cells

Character Cell Wall Prokaryotes Present Eukaryotes Animals & Protozoans Absent Plants, Fungi & Algae Present Cellulose or chitin

Composition

Peptidoglycan complex carbohydrate Flagella

Locomotor organelles

Flagella/ Cilia

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Prokaryotic Cells
Much smaller (microns) and more simple than eukaryotes Prokaryotes are molecules surrounded by a membrane and cell wall. They lack a true nucleus and dont have membrane bound organelles like mitochondria, etc. Large surface-to-volume ratio : nutrients can easily and rapidly reach any part of the cells interior
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Size of Bacteria
Unit of measurement in bacteriology is the micron (micrometre, m) Bacteria of medical importance
0.2 1.5 m in diameter 3 5 m in length
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Eukaryotic cell
(e.g. animal)
Rough endoplasmic reticulum Nucleus Cell membrane

Prokaryotic cell
Gram +
Flagellum Nucleoid Cell wall

Gram Pili

Granule

Cytoplasm
Mitochondria

Capsule Cell (inner) membrane Outer membrane Dr.T.V.Rao MD Ribosomes 18 Cell wall

Shapes of Bacteria
Cocci spherical/ oval shaped major groups Bacilli rod shaped Vibrios comma shaped Spirilla rigid spiral forms Spirochetes flexible spiral forms Actinomycetes branching filamentous bacteria Mycoplasmas lack cell wall
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Bacteria Have One of Three Cellular Shapes

Rods (bacilli)

Coccoid-Shaped

Spirilla

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Arrangement of bacteria: Cocci

Coccus Cocci in pair Diplococcus Tetrad groups of four

Cocci in chain - Streptococci

Cocci in cluster - Staphylococci

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Sarcina groups of eight

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Reproduction
Prokaryotic cell division is binary fission.
Single DNA molecule that first replicates. Attaches each copy to a different part of the cell membrane. Cell begins to pull apart. Following cytokinesis, there are then two cells of identical genetic composition.

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Arrangement of bacteria: Bacilli

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Other shapes of bacteria

Comma shaped

Spirilla

Spirochetes

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Anatomy of a Bacterial Cell

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Anatomy of A Bacterial Cell

Outer layer two components:
1. 2. Rigid cell wall Cytoplasmic (Cell/ Plasma) membrane present beneath cell wall

Cytoplasm cytoplasmic inclusions, ribosomes, mesosomes and nucleus Additional structures plasmid, slime layer, capsule, flagella, fimbriae (pili), spores
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Structure of Bacteria
All cells have 3 main components:
DNA (nucleoid)
genetic instructions

surrounding membrane (cytoplasmic membrane)

limits access to the cells interior

cytoplasm, between the DNA and the membrane

where all metabolic reactions occur especially protein synthesis, which occurs on the ribosomes

Bacteria also often have these features:

cell wall
resists osmotic pressure

flagella
movement

pili
attachment

capsule
protection and biofilms
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Typical shapes of bacteria

Most bacteria retain a particular shape; a few are pleiomorphic

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Typical prokaryotic structures

Working from the outside in

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Characteristic grouping (or not grouping)

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The Cell Envelope

Gram Positive

Gram Negative

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Lipoteichoic acid

GRAM POSITIVE
Peptidoglycan-teichoic acid

Cytoplasmic membrane

Cytoplasm

GRAM NEGATIVE
Porin

Lipopolysaccharide

Outer Membrane

Braun lipoprotein

Inner (cytoplasmic) membrane

Cytoplasm
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Gram-positive and gram-negative bacteria

Gram-Negative Bacteria

Difference Between Gram-Negative and Gram-Positive Bacteria

Gram-Positive Bacteria Simple cell wall.

More complex cell wall.

Thin peptidoglycan celll wall layer.

Thick peptidoglycan celll wall layer.

Outer lipopolysaccharide wall layer.

No outer lipopolysaccharide wall layer.

Retain safranin.

Retain crystal violet/iodine.

Appear pink/red.

Appear blue/purple.

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Cell Envelope
The cell envelope is all the layers from the cell membrane outward, including the cell wall, the periplasmic space, the outer membrane, and the capsule. All free-living bacteria have a cell wall periplasmic space and outer membrane are found in Gram-negatives the capsule is only found in some strains
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Structure & Function of Cell Components

CELL WALL
Outermost layer, encloses cytoplasm
1. Confers shape and rigidity 2. 10 - 25 nm thick
1. Composed of complex polysaccharides (peptidoglycan/ mucopeptide) - formed by N acetyl glucosamine (NAG) & N acetyl muramic acid (NAM) alternating in chains, held by peptide chains.
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Cell Wall
Cell wall

4. Carries bacterial antigens important in virulence & immunity 5. Chemical nature of the cell wall helps to divide bacteria into two broad groups Gram positive & Gram negative 6. Gram +ve bacteria have simpler chemical nature than Gram ve bacteria. 7. Several antibiotics may interfere with cell wall synthesis e.g. Penicillin, Cephalosporins
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Transport Across the Cell Membrane

Basic rule: things spontaneously move from high concentration to low concentration (downhill). This process is called diffusion.
Getting many molecules into the cell is simply a matter of opening up a protein channel of the proper size and shape. The molecules then move into the cell by diffusing down the concentration gradient. Passive transport, or facilitated diffusion.

To get things to move from low to high (uphill), you need to add energy: the molecules must be pumped into the cell. Pumps are driven by ATP energy. Dr.T.V.Rao MD Active transport.

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Outer Membrane
Gram negative bacteria

major permeability barrier space between inner and outer membrane Periplasmic space store degradative enzymes Gram positive bacteria no Periplasmic space
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Gram positive cell wall

The Gram-positive cell wall is composed of a thick, multilayered peptidoglycan sheath outside of the cytoplasmic membrane. Teichoic acids are linked to and embedded in the peptidoglycan, and lipoteichoic acids extend into the cytoplasmic membrane
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Gram negative cell wall

The Gram-negative cell wall is composed of an outer membrane linked to thin, mainly single-layered peptidoglycan by lipoproteins. The peptidoglycan is located within the periplasmic space that is created between the outer and inner membranes. The outer membrane includes porins, which allow the passage of small hydrophilic molecules across the membrane, and lipopolysaccharide molecules that extend into extracellular space.
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Cell Wall

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Summary of the differences between Gram positive & Gram negative bacteria
Property of bacteria Gram Positive

Gram Negative

Thickness of wall Number of layers in wall Peptidoglycan content Teichoic acid in wall Lipid & lipoprotein content Protein content Lipopolysaccharide Sensitive to penicillin Digested by lysozyme

20-80 nm 1 >50% + 0-3% 0% 0 Yes Yes

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10 nm 2 10-20% 58% 9% 13% Less sensitive Weakly

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Cytoplasmic (Plasma) membrane

Thin layer 5-10 nm, separates cell wall from cytoplasm Acts as a semipermeable membrane: controls the inflow and outflow of metabolites Composed of lipoproteins with small amounts of carbohydrates
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Other Cytoplasmic Components

Ribosomes protein synthesis Mesosomes
1. Multilaminated structures formed as invaginations of plasma membrane 2. Principal sites of respiratory enzymes 3. Coordinate nuclear & cytoplasmic division during binary fission 4. More prominent in Gram +ve bacteria

Intracytoplasmic inclusions reserve of energy & phosphate for cell metabolism e.g. Metachromatic granules in diphtheria bacilli
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Nucleus
No nucleolus No nuclear membrane Genome
single, circular double stranded DNA. Haploid Divides by binary fission
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Additional Organelles
1. Plasmid

Extra nuclear genetic elements consisting of DNA Transmitted to daughter cells during binary fission May be transferred from one bacterium to another Not essential for life of the cell Confer certain properties e.g. drug resistance, toxicity
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Additional Organelles
2.

Capsule & Slime layer

Viscous layer secreted around the cell wall. Polysaccharide / polypeptide in nature

a) Capsule sharply defined structure, antigenic in nature

Protects bacteria from lytic enzymes Inhibits phagocytosis Stained by negative staining using India Ink Can be demonstrated by Quellung reaction (capsule Dr.T.V.Rao MD swelling reaction)

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Additional Organelles
3. Flagella Long (3 to 12 m), filamentous surface appendages Organs of locomotion Chemically, composed of proteins called flagellins The number and distribution of flagella on the bacterial surface are characteristic for a given species - hence are useful in identifying and classifying bacteria Flagella may serve as antigenic determinants (e.g. the H antigens of Gram-negative enteric bacteria) Presence shown by motility e.g. hanging drop preparation Dr.T.V.Rao MD 50

Types of flagellar arrangement

Polar/ Monotrichous single flagellum at one pole

Lophotrichous tuft of flagella at one pole

Amphitrichous flagella at both poles

Peritrichous flagella all over

Amphilophotrichous tuft of flagella at both ends

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Cocci do not have flagella

Peritrichous (or amphi, or

monotrichous lophotrichous
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FLAGELLA
Some bacteria are motile Locomotory organelles- flagella Taste environment Respond to food/poison chemo taxis
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 Flagella embedded in cell membrane project as strand Flagellin (protein) subunits move cell by propeller like action

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Additional Organelles
4.

Fimbriae/ Pili
Thin, hairlike appendages on the surface of many Gramnegative bacteria 10-20 long, acts as organs of adhesion (attachment) - allowing
bacteria to colonize environmental surfaces or cells and resist flushing

Made up of proteins called pilins.

Pili can be of two types

Common pili short & abundant Sex pili - small number (one to six), very long pili, helps in conjugation (process of transfer of DNA)
http://student.ccbcmd.edu/courses/bio141/lecguide/unit1/prostruct/yespili.html
http://student.ccbcmd.edu/courses/bio141/lecguide/unit1/prostruct/nopili.html
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Additional Organelles
5. Spores Highly resistant resting stages formed during adverse environment (depletion of nutrients) Formed inside the parent cell, hence called Endospores Very resistant to heat, radiation and drying and can remain dormant for hundreds of years. Formed by bacteria like Clostridia, bacillus
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The cycle of spore formation and germination

At the beginning of spore formation, a septum forms, separating the nascent spore from the rest of the cell and all of the genetic material of the cell is copied into the newly-forming cell. The spore contents are dehydrated and the protective outer coatings are laid down. Once the spore is matured it is l released from the cell. On germination, the spore contents rehydrate and a new bacterium emerges and multiplies.
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Shape & position of bacterial spore

Oval central Spherical central Oval sub terminal Oval sub terminal Bulging Non bulging

Oval terminal Spherical terminal Free spore

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Spores
Some bacteria can form very tough spores, which are metabolically inactive and can survive a long time under very harsh conditions.

Allegedly, some bacterial spores that were embedded in amber or salt deposits for 25 million years have been revived. These experiments are viewed skeptically by many scientists.
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Spores
Spores can also survive very high or low temperatures and high UV radiation for extended periods. This makes them difficult to kill during sterilization. Anthrax Spores are produced only by a few genera in the Firmicutes: Bacillus species including anthracis (anthrax) and cereus (endotoxin causes ~5% of food poisoning) Clostridium species including tetani (tetanus), perfringens (gangrene), and botulinum (botulism: food poisoning from improperly canned food)

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Pleomorphic & Involution forms

Pleomorphism great variation in shape & size of individual cells e.g. Proteus species Involution forms swollen & aberrant forms in ageing cultures, especially in the presence of high salt concentration e.g. plague bacillus Cause defective cell wall synthesis
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 Includes three components:

1. Classification : orderly arrangement 2. Identification of an unknown unit 3. Nomenclature : naming the units
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Bacterial Taxonomy

Bacterial Taxonomy: Classification

Orderly arrangement : Kingdom Division Class Order Family Tribe Genus Species Phylogenetic classification represents a branching tree like arrangement. One characteristic being used for division at each branch or level Molecular or Genetic classification based on the degree of genetic relatedness of different organisms Intraspecies classification based on biochemical properties (biotypes), antigenic features (serotypes), bacteriophage susceptibility (phage types)

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Bacterial Taxonomy: Nomenclature

Two kinds of name are given to bacteria

Casual / common name for local use, varies from country to country e.g. typhoid bacillus Scientific / International Name same all over world, consists of two words (in Italics) e.g. Salmonella typhi, Staphylococcus
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Microscopy helps to Measure and Observe the Bacteria

Measurement
Microorganisms are very small Use metric system Metre (m) : standard unit Micrometre (m) = 1 x10-6 m Nanometre (nm) = 1 x10-9 m Angstrom () = 1 x10-10 m
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Why we should be Stain Bacteria

Bacteria have nearly the same refractive index as water, therefore, when they are observed under a microscope they are opaque or nearly invisible to the naked eye. Different types of staining methods are used to make the cells and their internal structures more visible under the light microscope.
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What is a Stain
A stain is a substance that adheres to a cell, giving the cell color. The presence of color gives the cells significant contrast so are much more visible. Different stains have different affinities for different organisms, or different parts of organisms They are used to differentiate different types of organisms or to view specific parts of organisms
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 Methylene blue, Basic fuchsin Provide the color contrast but impart the same color to all the organisms in a smear Loffler's ethylene blue: Sat. solution of M. blue in alcohol - 30mlKoH, 0.01% in water 100mlDissolve the dye in water, filter. For smear: stain for 3. For section: stain
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Simple staining

Simple Staining Easier to Perform But has Limitations

Simple easy to use; single staining agent used; using basic and acid dyes. Features of dyes: give coloring of microorganisms; bind specifically to various cell structures

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Differential Stains
Differential Stains use two or more stains and allow the cells to be categorized into various groups or types. Both techniques allow the observation of cell morphology, or shape, but differential staining usually provides more information about the characteristics of the cell wall (Thickness).
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Gram staining
Named after Hans Christian Gram, differentiates between Grampositive purple and Gram-negative pink stains and is used to identify certain pathogens.
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Gram staining - Principles

Gram staining is used to determine gram status to classify bacteria broadly. It is based on the composition of their cell wall. Gram staining uses crystal violet to stain cell walls, iodine as a mordant, and a fuchsin or safranin counterstain to mark all bacteria. Gram status is important in medicine; the presence or absence of a cell wall will change the bacterium's susceptibility to some antibiotics. Gram-positive bacteria stain dark blue or violet. Their cell wall is typically rich with peptidoglycan and lacks the secondary membrane and lipopolysaccharide layer found in Gram-negative bacteria
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Gram Staining Steps

1. Crystal violet acts as the primary stain. Crystal violet may also be used as a simple stain because it dyes the cell wall of any bacteria. 2. Grams iodine acts as a mordant (Helps to fix the primary dye to the cell wall). 3. Decolorizer is used next to remove the primary stain (crystal violet) from Gram Negative bacteria (those with LPS imbedded in their cell walls). Decolorizer is composed of an organic solvent, such as, acetone or ethanol or a combination of both.) 4. Finally, a counter stain (Safranin), is applied to stain those cells (Gram Negative) that have lost the primary stain as a result of decolorization
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Structure and Reactivity to Gram Staining.

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Gm+ve cocci & Gm-ve bacilli

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GRAM-POSITIVE BACTERIA
GRAM-POSITIVE BACTERIA are characterized by having as part of their cell wall structure peptidoglycan as well as polysaccharides and/or teichoic acids. The peptidoglycans which are sometimes also called murein are heteropolymers of glycan strands, which are cross-linked through short peptides.

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What are Gram Negative Bacteria

Gram-negative bacteria are those bacteria that do not retain crystal violet dye in the Gram staining protocol. In a Gram stain test, a counter stain (commonly safranin) is added after the crystal violet, coloring all Gram-negative bacteria with a red or pink color. The test itself is useful in classifying two distinct types of bacteria based on the structural differences of their cell walls. On the other hand, Gram-positive bacteria will retain the crystal violet dye when washed in a decolorizing solution.
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Gram negative bacteria

On most Gram-stained preparations, Gramnegative organisms will appear red or pink because they are counterstained. Due to presence of higher lipid content, after alcoholtreatment, the porosity of the cell wall increases, hence the CVI complex (Crystal violet -Iodine) can pass through. Thus, the primary stain is not Dr.T.V.Rao MD retained.

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Gram Negative Bacteria

Also, in contrast to most Gram-positive bacteria, Gram-negative bacteria have only a few layers of peptidoglycan and a secondary cell membrane made primarily of lipopolysaccharide

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ACID FAST STAINING

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Acid-Fast Stain
Acid-fast cells contain a large amount of lipids and waxes in their cell walls

primarily mycolic acid

Acid fast bacteria are usually members of the genus Mycobacterium or Nocardia
Therefore, this stain is important to identify Mycobacterium or Nocardia

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Ziehl-Neelsen stain
Ziehl-Neelsen staining is used to stain species of Mycobacterium tuberculosis that do not stain with the standard laboratory staining procedures like Gram staining. The stains used are the red colored Carbol fuchsin that stains the bacteria and a counter stain like Methylene blue or Malachite green.
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Acid-Fast Organisms
Primary stain binds cell wall mycolic acids Intense decolorization does not release primary stain from the cell wall of AFB Color of AFB-based on primary stain Counterstain provides contrasting background

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AFB Staining Methods

Zeihl Neelsens-hot stain Kinyouns-cold stain Modifications
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ALBERTS STAINING FOR C.diptheria

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Diphtheria is Serious Disease When you suspect Diphtheria

In all cases of suspected cases of Diphtheria, stain one of the smears with Gram stain If Gram stained smear shows morphology suggestive of C.diptheria, proceed to do Albert staining which demonstrates the presence or absence of metachromatic granules.
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Appearance of C.diptheria
C.diptheria are thin Gram positive bacilli, straight or slightly curved and often enlarged (clubbing) at one or both ends and are arranged at acute angles giving shapes of Chinese letters or V shape which is characteristic of these organisms (Fig 1). Present in the body of the bacillus are numerous metachromatic granules which give the bacillus beaded or barred appearance. These granules are best demonstrated by Alberts stain.
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Albert staining
Albert stain I Toluidine blue 0.15 gm Malachite green 0.20 gm Glacial acetic acid 1.0 ml Alcohol(95%) 2.0 ml Distilled water 100 ml Albert stain II

Iodine 2.0 gm Potassium iodide 3.0 gm Distilled water 300 ml

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Albert staining Procedure

Cover the heat-fixed smear with Albert stain I. Let it stand for two minutes. Wash with water. Cover the smear with Albert stain II. Let it stand for two minutes. Wash with water, blot dry and examine.
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How the C.diptheria appear

To demonstrate metachromatic granules in C.diptheria. These granules appear bluish black whereas the body of bacilli appear green or bluish green.

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 Programme created by Dr.T.V.Rao MD from several resources in world wide web, and Thankful for Dr. Ekta www.medmicrobes from basic programme on Bacterial cell Email
doctortvrao@gmail.com
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