An introduction An introduction into into

MicroMicrobiobiologylogy

Dr. Majid Zare Bidaki(PhD in Medical Microbiology, Assistant professor)

Birjand University of Medical Science

ReferencesReferences

1. Jawetz, Melnick, & Adelberg; Medical Microbiology, 24th ed.

2. Zinsser, Hans, And Joklik, Wolfgang K.; Medical Microbiology, 17th ed.

3. Baron, Samuel; Medical Microbiology, 4th ed.

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Antonie van LeeuwenhoekAntonie van Leeuwenhoek

First to observe living microbes

his single-lens magnified up to 300X

(1632-1723)

4

5

Louis Pasteur (1822-1895) Louis Pasteur (1822-1895)

Showed microbes caused fermentation & spoilage

Disproved spontaneous generation of microbes

Developed aseptic techniques.

Developed a rabies vaccine.

6

Robert Koch Robert Koch

Proposed: Germ theory of disease

Developed: pure culture methods.

Identified: cause of anthrax, TB, & Cholera.

(1843-1910)

Acellular and cellular Microorganisms

Acellular: VirusesViruses ViroidsViroids PrionsPrions

Cellular:BacteriaBacteriafungifungiProtista: Protozoa & algaeProtista: Protozoa & algaehelminths (worms) helminths (worms)

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Prokaryotic or EukaryoticProkaryotic or Eukaryotic

Prokaryotes vs EukaryotesProkaryotes vs Eukaryotes

Size smaller Larger

Nucleus - +

Organelles - +

Chromosomes 1 circular Multiple, linear

Ribosomes smaller 70s Larger 80sr

In prokaryotes against Eukaryotes, cell membranes lack In prokaryotes against Eukaryotes, cell membranes lack sterols (e.g. cholesterol)sterols (e.g. cholesterol)

Living OrganismsLiving OrganismsClassificationClassification

• Domain

• Kingdom

• Phylum

• Class

• Order

• Genus

• Species

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Scientific nomenclatureScientific nomenclature

Binomial (scientific) nomenclature Gives each microbe 2 namesGenusGenus - noun, always capitalizedspeciesspecies - adjective, lowercase

Both italicized or underlined– Staphylococcus aureus (S. aureus)– Escherichia coli (E. coli)

Bergey's Manual of Systematic Bacteriology

Shapes of BacteriaShapes of Bacteria

Shapes of BacteriaShapes of Bacteria

Bacterial shapesBacterial shapes

Size of Bacteria

Gram positiveGram positive Gram negativeGram negative

LipopolysaccharideLipopolysaccharide

Lipid A• Glucosamine disaccharide• Beta hydroxy fatty acids

Core • Heptoses• Ketodeoxyoctonic acid

O-antigenHighly variable

(Hydroxy myritic Acid)

LPS functionLPS functionEndotoxinsEndotoxinsExotoxinsExotoxins

PeptidoglycanPeptidoglycan

Cytoplasmic membrane Cytoplasmic membrane ProtoplastProtoplast

SpheroplastSpheroplast L formsL forms

Gram positiveGram positive

Gram negativeGram negative

4 groups based on cell wall 4 groups based on cell wall compositioncomposition

1. Gram positive cells

2. Gram negative cells

3. Bacteria without cell walls

4. Bacteria with chemically unique cell walls

Gram positive wall

Gram negative cell wallGram negative cell wall

CytoplasmCytoplasm

CytoplasmCytoplasm

Lipoteichoic acidPeptidoglycan-teichoic acid

Cytoplasmic membrane

Inner (cytoplasmic) membrane

Outer Membrane

LipopolysaccharidePorin

lipoprotein

Peri

plas

mic

spa

ce

r r rrr

r

Gram Positive Cell EnvelopeGram Positive Cell Envelope

rrrr

Lipoteichoic acidPeptidoglycan-teichoic acid

Cytoplasmic membrane

Bacteria classification based on Bacteria classification based on cell wall structurecell wall structure

Grasilicutes (Gram Negative)Grasilicutes (Gram Negative)Firmicutes (Gram Positive)Firmicutes (Gram Positive)Tenricutes (with no Cell wall)Tenricutes (with no Cell wall)Mendosicutes (with no Peptidoglycan in Mendosicutes (with no Peptidoglycan in

cell wall)cell wall)

Major Taxonomic Groups of BacteriaMajor Taxonomic Groups of Bacteria

Gracilicutes – gram-negative cell walls, thin-skinned

Firmicutes – gram-positive cell walls, thick skinned

Tenericutes – lack a cell wall & are softMendosicutes – archaea, primitive procaryotes

with unusual cell walls & nutritional habits

CapsuleCapsule

2 types1. Macro capsule - highly organized, tightly

attached2. Micro capsule, Slime layer or Glycocalyx -

loosely organized and attached

Functions attachment inhibits killing by white blood cells Receptor (K antigen)

2 Types of Capsule2 Types of Capsule

BiofilmsBiofilms

FlagellaFlagella

Fimbrea (Pili)Fimbrea (Pili)

Adhesion to other cells and surfaces

StructureStructure

Pili & Sex pili

Rigid tubular structure made of pielin protein

Found mostly in Gram negative cells

FunctionsFunctions Adhesionjoins bacterial cells for DNA transfer (Conjugation)

ConjugationConjugation

CytoplasmCytoplasmDense gelatinous solution of sugars, amino

acids, & salts70-80% waterServes as solvent for materials used in all

cell functions

ChromosomeChromosome

single, circular, double-stranded DNA molecule that contains all the genetic information required by a cell

DNA is tightly coiled around a protein, aggregated in a dense area called the nucleoid

plasmidsplasmids

small circular, double-stranded DNA

free or integrated into the chromosome

duplicated and passed on to offspring

not essential to bacterial growth & metabolism

may encode antibiotic resistance, tolerance to toxic metals, enzymes

& toxins

used in genetic engineering- readily manipulated & transferred

from cell to cell

RibosomesRibosomes

made of 60% ribosomal RNA & 40% protein

consist of 2 subunits: large (50 S) & small (30 S)

procaryotic differ from eucaryotic ribosomes in

size & number of proteins

site of protein synthesis

All bacterial cells have ribosomes.

Inclusions, granulesInclusions, granules

intracellular storage bodies

vary in size, number & content

bacterial cell can use them when

environmental sources are depleted

Examples: glycogen, sulfur and polyphosphate granules,

poly-b-hydroxybutyrate, gas vesicles for floating.

EndosporesEndospores

Important components in endospore: Important components in endospore:

CalciumCalciumDipicolinic AcidDipicolinic Acid

Endospore structureEndospore structure

Spore structureSpore structure

Spherical or Oval

Terminal, subterminal or central

Bulging or nobulging

Growth in BacteriaGrowth in Bacteria

Temperature

Nutrients

pH

Osmotic pressure

Minimum temperature – lowest temperature that

permits a microbe’s growth and metabolism

Maximum temperature – highest temperature

that permits a microbe’s growth and metabolism

Optimum temperature – promotes the fastest

rate of growth and metabolism

Temperature Temperature

3 temperature adaptation groups

Bacterial MetabolismBacterial Metabolism

• DefinitionDefinition

• RequirmentsRequirments: ? & ?? & ?

Phototroph

Chemotroph

Autotroph

Heterotroph

Bacterial MetabolismBacterial Metabolism

PhototrophPhotoautotroph (Photolitotroph) Photoheterotroph (Photoorganotroph)

ChemotrophChemoautotroph (ChemolitotrophChemoheterotroph (Chemoorganotroph)

Stages of metabolism in Stages of metabolism in chemoheterotrophic bacteriachemoheterotrophic bacteria

Digestion

Absorption (Passive and active transportation)

Preparation for oxidation

Oxidation

Oxidation & ReductionOxidation & Reduction

X Ye- & H+ Cytochromes, …. Cytochromes, ….

Aerobics, Fermentors, Anaerobics

• In Aerobics:

Last e- receptor is Oxigen.

• In Fermentors:

Last e- receptor is an organic material.

• In Anaerobics:

Last e- receptors is a mineral material.

Oxygen requirementsOxygen requirements

Bacterial growth in closed & Bacterial growth in closed & continuous culturescontinuous cultures

• Closed culture:

There is no chance for adding or reducing the requirements or wastes.

• Continuous culture:

Fresh requirements come in and wastes goes out continuously.

The curve of bacterial growth in a closed The curve of bacterial growth in a closed cultureculture

Growth Curve

Bacterial growth in a Bacterial growth in a continuous culturecontinuous culture

Con

tinuo

us C

ultu

re, C

hem

osta

t

FermentersFermenters

Bacterial growth Bacterial growth

Binary divisionBinary division

Microbial growth calculationMicrobial growth calculation

b = a.2n

G (Generation time) = T / n (n = The number of generations, T = The total time of growth for the population)

Measuring the bacterial growthMeasuring the bacterial growth

• Measuring the number of bacteria- Turbidity- Cell counting• Measuring the mass of bacteria

Measuring the number of bacteriaMeasuring the number of bacteria

• Torbidity (light absorbtion)Torbidity (light absorbtion)

- Measuring with eye

- Measuring with devices

Measuring with devices

Cell counting(by device)

Cell counting(by Microscopy)

Cell counting(by culture)

FermentationFermentation

• Defined as an energy yielding process

• Organic molecules serve as both electron donors and electron accepters.

• The molecule being metabolized does not have all its potential energy extracted from it. (not completely oxidized.)

• NAD+ is almost always reduced to NADH

• Remember that metabolism involves the oxidation of the substrate. These electrons are removed from the organic molecule and most often given to NAD. (This is true both in fermentation and respiration).

• Fermentation results in a excess of NADH

• Accumulation of NADH causes a problem for anaerobes. They have too much of it and it prevents further oxidation of substrate due to a lack of an NAD+ pool to accept electrons.

• In many fermentation pathways, the steps after energy generation are performed in part to get rid of the NADH:

pyruvate + NADH + H+ → lactate + NAD+

Glycolysis - Embden-Meyerhoff-Parnas pathway (EMP)

• The most commonly used series of reactions for oxidizing glucose glucose toto pyruvate pyruvate.

• EMP is so ubiquitous. However, it is not the only method for the fermentation of glucose.

• It is an essential part of many organisms catabolism.

• A complete fermentation pathway begins with a substrate, includes glycolysisglycolysis and results in various end-productsvarious end-products.

• The different fermentation pathways typically are named for the end products that are formed.

• yeast, convert NADH back to NAD+ in a process called ethanol fermentation. In this process, the pyruvate pyruvate is converted first to acetaldehyde acetaldehyde andand carbon dioxide carbon dioxide, then to ethanolethanol.

PyruvatePyruvate

• Pyruvate is often an important intermediate

• Many of the reactions that we will look at eventually end up making pyruvate.

• Pyruvate is a valuable intermediate because it can be used for cell synthesis.

• Many different enzymes can act on it. It gives the microbe flexibility.

Fermentation Vs Aerobic oxidation

.C6H12O6------------>2C2H5OH + 2CO2 (2ATP) ∆G = -

33 Kcal تخمیر

 C6H12O6 + 6O2------------>6CO2 + 6H2O (36ATP) ∆G =

686 Kcal_تنفس

FermentationFermentation

Incomplete oxidation of glucose or other carbohydrates in the absence of oxygen

Uses organic compounds as terminal electron acceptors

Yields a small amount of ATP

Production of ethyl alcohol by yeasts acting on glucose

Formation of acid, gas & other products by the action of various bacteria on pyruvic acid

GlycolysisGlycolysis

Embden-Meyerhof-Parnas Embden-Meyerhof-Parnas (EMP pathway)(EMP pathway)

Entner–Doudoroff pathwayEntner–Doudoroff pathway

• The Entner–Doudoroff pathway describes an alternate series of reactions that catabolize glucose to pyruvate using a set of enzymes different from those used in either glycolysis or the pentose phosphate pathway

Homolactic FermentationHomolactic Fermentation ((Lactic acid)Lactic acid)

- It is the sole end product.

- Pathway of the homolactic acid bacteria (Lactobacillus, Lactococcus and most streptococci).

- The bacteria are used to ferment milk and milk products in the manufacture of yogurt, buttermilk, sour cream, cottage cheese, cheddar cheese….

Mixed Acid FermentationsMixed Acid Fermentations..• Mainly the pathway of the Enterobacteriaceae.

• End products are a mixture of lactic acid, acetic acid, formic acid, succinate and ethanol.

• Possibility of gas formation (CO2 and H2) if the bacterium possesses the enzyme formate formate dehydrogenasedehydrogenase, which cleaves formate to the gases.

• Mixed acids and gases but in addition, butanediol from the condensation of 2 pyruvate. The use of the pathway decreases acid formation (butanediol is neutral) and causes the formation of a distinctive intermediate, acetoin.

• Specific tests to detect low acid and acetoin in order to distinguish non fecal enteric bacteria (butanediol formers, such as Klebsiella and Enterobacter) from fecal enterics (mixed acid fermenters, such as E. coli, Salmonella and Shigella).

Butanediol FermentationButanediol Fermentation

Butyric acid fermentationsButyric acid fermentations((Butanol-acetone fermentation)

• Butyric acid Butyric acid fermentations as well as the butanol-butanol-acetoneacetone fermentation, are run by the clostridia, the masters of fermentation.

• In addition to butyric acid, the clostridia form acetic acid, CO2 and H2 from the fermentation of sugars. Small amounts of ethanol and isopropanol may also be formed.

• Butanol and acetone were discovered as the main end products of fermentation by Clostridium acetobutylicum

• Carried out by the propionic acid bacteria which include corynebacteria, Propionibacterium and Bifidobacterium.

• Propionic acid bacteria ferment lactate to acetic acid, CO2 and propionic acid.

• The formation of propionate is a complex and indirect process involving 5 or 6 reactions.

• The propionic acid bacteria are used in the manufacture of Swiss cheeseSwiss cheese, which is distinguished by the distinct flavor of propionate and acetate, and holes caused by entrapment of CO2.

Propionic acid fermentation.

FermentationFermentation

Fermentations in bacteria that proceed through the Embden-Meyerhof pathway. Representive bacteria that utilize these pathways

are in shown in BLUE.

MediumMedium

DefinitionDefinitionTypes based on solidity:Types based on solidity:

1. Liquid medium

BHI, TSB, SF, NB, …

2. Solid medium

Blood agar, Nutrient agar, chocolate agar, Columbia agar, EMB

3. Semi-solid medium

SIM

•A Undefined mediumUndefined medium (a basal or complex medium). Contains: - A source of amino acids and nitrogen (e.g., beef, yeast extract)- A carbon source such as glucose for bacterial growth- water- Various salts needed for bacterial growth

•A defined mediumdefined medium (a synthetic medium) is a medium in which- all the chemicals used are known- no yeast, animal or plant tissue is present

Nutrient media (Broth/Agar)

Nutrient agar medium composition:Nutrient agar medium composition: - Beef Extract-0.3gm (mineral and carbohydrate) - Peptone-0.5gm (protein and nitrogen source) - NaCl-0.5gm (electrolyte) - Agar-1.5gm (solidifying agent) - Distilled water-100ml pH-7

Minimal media

contain the minimum nutrients possible for colony growth, generally without the presence of amino acids.used to grow "wild type" microorganisms and select for or against recombinants.

Minimal medium typically contains:Minimal medium typically contains:-A carbon source carbon source (such as glucose, or a less energy-rich source like succinate.- various saltssalts (provide essential elements such as magnesium, nitrogen, phosphorus, and sulfur to allow the bacteria to synthesize protein and nucleic acid- WaterWater

Supplementary minimal media

• A type of minimal media that also contains a single selected agent, usually an amino acid or a sugar.

• This supplementation allows for the culturing of specific lines of auxotrophic recombinants.

Culture mediaCulture mediabased on the application in clinical labsbased on the application in clinical labs

General medium (Nutrient agar, Muller hinton agar, Nutrient broth, …)

Selective/Special medium (EMB, MSA, …)

Differential medium (MAC [MacConkey agar], EMB, XLD, …)

Enrichment medium (SF, RV [Rappaport-Vassiliadis Broth], TT, …)

Enriched medium (Blood agar, Chocolate agar, cold enrichment)

Transport medium (Stuart, Cary-Blair, Amies, …)

Cary-Blair: exclusively associated with enteric transport

Stuart & Amies: widely used for the transportation of a diverse range of clinical swab samples from sites including the eye,

ear, nose, throat, skin, genital tract and wounds.

Galleries (TSI, SIM, MRVP, Citrate agar, ….)

General mediumGeneral medium

• Theoretically it supports cultivation of many bacteria.

• Nutrient agar, Muller hinton agar, Nutrient broth, …

Selective/Specialized medium• Some examples of selective media include:

• Eosin methylene blue (EMB) that contains methylene blue – toxic to Gram-positive bacteria, allowing only the growth of Gram negative bacteria

• YM (yeast and mold) which has a low pH, deterring bacterial growth

• MacConkey agar for Gram-negative bacteria

• brilliant green agar, a medium that inhibits Gram-positive bacteria while permitting Gram-negative organisms such as Salmonella species to grow.

Selective/Special medium• Hektoen enteric agar (HE) which is selective for Gram-

negative bacteria

• Mannitol salt agar (MSA) which is selective for Gram-positive bacteria and differential for mannitol

• Terrific Broth (TB) is used with glycerol in cultivating recombinant strains of Escherichia coli.

• xylose lysine desoxyscholate (XLD), which is selective for Gram-negative bacteria

• Buffered charcoal yeast extract agar, which is selective for certain gram-negative bacteria, especially Legionella pneumophila

Differential media• Blood agar which contains bovine heart blood that

becomes transparent in the presence of hemolytic streptococcus.

• Eosin methylene blue (EMB), which is differential for lactose and sucrose fermentation.

• MacConkey (MCK), which is differential for lactose fermentation.

• Mannitol salt agar (MSA), which is differential for mannitol fermentation.

• violet red bile agar is used to distinguish coliform bacteria from noncoliform organisms.

Transport Media

• Maintain the viability Maintain the viability of organisms in specimen.• No altering No altering microorganism concentration.• Contain only buffersbuffers and saltsalt. • Lack of carbon, nitrogen, and organic Lack of carbon, nitrogen, and organic growth

factors.• Transport media used in the isolation of anaerobesanaerobes

must be free of molecular oxygenfree of molecular oxygen.

Transport Media

• ThioglycolateThioglycolate brothbroth for restrict anaerobes.• Stuart transport medium Stuart transport medium - a non-nutrient soft agar

gel containing a reducing agent to prevent oxidation, and charcoal to neutralise.

• Certain bacterial inhibitors- for gonococci, and buffered glycerol saline for enteric bacilli.

• Venkat-Ramakrishnan(VR) medium for v. cholerae.

• Some organisms, termed fastidious organisms, require specialized environments due to complex nutritional requirements.

Methods in bacterial identificationMethods in bacterial identification

1. Microscopic morphology

2. Macroscopic morphology – colony appearance

3. Physiological / biochemical characteristics

4. Chemical analysis

5. Serological analysis

6. Genetic & molecular analysis G + C base composition DNA analysis using genetic probes Nucleic acid sequencing & rRNA analysis

Bacterial ColoniesBacterial Colonies

• Standard Bacterial Count

• Colony-Forming Units

• Plaque-Forming Units

•Spread Plate

• Pour Plate

• Soft-Agar Overlay

Types of culture methodsTypes of culture methods

Isolation culture

Spread culture

Pour plate culture

Colony count culture