BacteriologyBasics Morphology, Classification, Staining

MethodsDr.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)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). 4

Dr.T.V.R

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The Light Microscope• many types

–bright-field microscope–dark-field microscope–phase-contrast microscope–fluorescence microscopes

• compound microscopes– image formed by action of 2 lenses 5

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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.

– Eyepiece or Ocular Lens: the lens closest to the eye.• Monocular: a microscope having only one eyepiece• Binocular: a microscope having two eyepieces. 6

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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 transparentobjects as dark on lightbackground (without staining)

• Right; human brain glialcells Dr.T.V.Rao MD 7

The Bright-Field Microscope• Produces a dark image against a

brighter background• Has several objective lenses

–par focal microscopes remain in focus when objectives are changed

• total magnification– product of the magnifications of the

ocular lens and the objective lens 8

Dr.T.V.R

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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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Dr.T.V.Rao MD

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 Earth’s 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 cellsCharacter Prokaryotes Eukaryotes

Nucleus Nuclear membrane

Absent Present

Nucleolus Absent Present

Chromosome One circular One or more paired and linear

Cell division Binary fission Mitosis

Cytoplasmic membrane

Structure and Composition

fluid phospholipid bilayer, lacks sterols

fluid phospholipid bilayer containing sterols

Function Incapable of endocytosis (phagocytosis and pinocytosis) and exocytosis

Capable of endocytosis and exocytosis

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Differences between prokaryotic & eukaryotic cells

Character Prokaryotes Eukaryotes

Cytoplasm Mitochondria Absent Present

Lysosomes Absent Present

Golgi apparatus Absent Present

Endoplasmic reticulum

Absent Present

Vacuoles Absent Present

Ribosomes 70 S 80 SDr.T.V.Rao MD 14

Differences between prokaryotic & eukaryotic cells

Character Prokaryotes Eukaryotes

Cell Wall Present Animals & Protozoans – AbsentPlants, Fungi & Algae - Present

Composition Peptidoglycan – complex carbohydrate

Cellulose or chitin

Locomotor organelles

Flagella 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 don’t 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, µm)

• Bacteria of medical importance–0.2 – 1.5 µm in diameter–3 – 5 µm in length

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Eukaryotic cell Prokaryotic cellGram +

Gram -

Cell wallRough endoplasmic reticulum

Mitochondria

(e.g. animal)Nucleoid

Nucleus

Cell membrane

Cytoplasm

Flagellum

Cell (inner) membrane Outer membraneRibosomes

Granule

Cell wall

Capsule

Pili

Dr.T.V.Rao MD

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

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Cocci in pair – Diplococcus

Sarcina – groups of eight

Tetrad – groups of four

Cocci in chain - Streptococci

Cocci in cluster - Staphylococci

Coccus

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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

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Comma shaped

Spirochetes

Spirilla

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Anatomy of a Bacterial Cell

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Anatomy of A Bacterial Cell• Outer layer – two components:

1. Rigid cell wall2. 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 cell’s 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 feware 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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GRAM POSITIVE

GRAM NEGATIVECytoplasm

Cytoplasm

Lipoteichoic acid Peptidoglycan-teichoic acid

Cytoplasmic membrane

Inner (cytoplasmic) membrane

Outer Membrane

LipopolysaccharidePorin

Braun lipoprotein

Perip

las m

ic sp

ace

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Gram-positive and gram-negative bacteria

Difference Between Gram-Negative

and Gram-Positive BacteriaGram-Negative Bacteria Gram-Positive Bacteria

More complex cell wall. Simple 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 rigidity2. 10 - 25 nm thick

3. 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. Active transport. Dr.T.V.Rao MD 39

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Outer Membrane Gram negative bacteria

• major permeability barrier • space between inner and outer

membrane–Periplasmic spacestore degradative enzymes

• Gram positive bacteria • no Periplasmic space

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Gram positive cell wall

Dr.T.V.Rao MD

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

Dr.T.V.Rao MD

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

Dr.T.V.Rao MD

Property of bacteria Gram Positive Gram Negative

Thickness of wall 20-80 nm 10 nm Number of layers in wall 1 2 Peptidoglycan content >50% 10-20% Teichoic acid in wall + - Lipid & lipoprotein content 0-3% 58% Protein content 0% 9% Lipopolysaccharide 0 13% Sensitive to penicillin Yes Less sensitive Digested by lysozyme Yes 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 enzymes3. Coordinate nuclear & cytoplasmic division during

binary fission4. 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 Organelles1. 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, toxicityDr.T.V.Rao MD 48

Additional Organelles2. 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 swelling reaction)

b) Slime layer – loose undemarcated secretion

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Additional Organelles3. Flagella –

– Long (3 to 12 µm), 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

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Types of flagellar arrangement

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Polar/ Monotrichous – single flagellum at one pole

Lophotrichous – tuft of flagella at one pole

Peritrichous – flagella all over

Amphitrichous – flagella at both poles

Amphilophotrichous – tuft of flagella at both ends

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Peritrichous monotrichous(or amphi, or lophotrichous

Cocci do not have flagella

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FLAGELLA

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• 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 Organelles4. Fimbriae/ Pili –

– Thin, hairlike appendages on the surface of many Gram-negative bacteria

– 10-20µ 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)

Dr.T.V.Rao MD

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 Organelles5. 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 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

Oval terminal

Spherical terminal

Free spore

Non bulging

Bulging

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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 synthesisDr.T.V.Rao MD 61

Bacterial Taxonomy• Includes three components:

1. Classification : orderly arrangement

2. Identification of an unknown unit

3. Nomenclature : naming the units

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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 aureus

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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 (Å) = 1 x10-10 m

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Why we should be Stain BacteriaBacteria 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..

66Dr.T.V.Rao MD

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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Simple staining • 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 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). 70Dr.T.V.Rao MD

Gram staining• Named after Hans

Christian Gram, differentiates between Gram-positive 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

72Dr.T.V.Rao MD

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. Gram’s 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.

77Dr.T.V.Rao MD

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.

78Dr.T.V.Rao MD

Gram negative bacteria• On most Gram-stained

preparations, Gram-negative organisms will appear red or pink because they are counterstained. Due to presence of higher lipid content, after alcohol-treatment, the porosity of the cell wall increases, hence the CVI complex (Crystal violet -Iodine) can pass through. Thus, the primary stain is not 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

82Dr.T.V.Rao MD

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. 83Dr.T.V.Rao MD

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

84Dr.T.V.Rao MD

AFB Staining Methods

• Zeihl Neelsen’s-hot stain

• Kinyoun’s-cold stain

• Modifications85Dr.T.V.Rao MD

ALBERT’S 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 Albert’s stain.

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Albert staining• Albert stain I• Toluidine blue 0.15 gm

Malachite green 0.20 gmGlacial acetic acid 1.0 mlAlcohol(95%) 2.0 mlDistilled water 100 ml

• Albert stain II

• Iodine 2.0 gmPotassium iodide 3.0 gmDistilled 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.

• 91Dr.T.V.Rao MD

• 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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