Bacte Prelim Lecnotes 2012

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Joanne Krystine G. Tago, RMT, MAST

MLS 302 – Microbiology 1



Introduction to Microbiology

Describe the historical development of

microbiology

Enumerate the development of science withemphasis on person/scientists and their

contributions

Explain the divisions of microbiology

Define terms



Historical Development: The beginnings… Robert Hooke

“Micrographia”

Compound microscope and its

uses

History of cell biology

Anton van Leeuwenhoek

First to observe livemicroorganisms (animalcules )

Single-lens microscope



Carolus Linnaeus

Binomial nomenclature : genus & specificepithet.

Are italicized or underlined.

Are “Latinized” and used worldwide.

May be descriptive or honor a scientist.

Staphylococcus aureus

Escherichia coli



Spontaneous Generation / Abiogenesis:

that living organisms arise from nonliving

matter

a “vital force” forms life.

Biogenesis:

that the living organisms arise from pre-existing life.

Abiogenesis versus Biogenesis



Aristotle’s hypothesis: .

Jan Baptista van Helmont’s _______________:

Decayingmaterial

livinganimals

dirtyshirt or

rags

a few grainsof wheat /wheat bran

+ =

Abiogenesis / Spontaneous Generation



Francesco Redi

1st real experiment to dispute abiogenesis

CONDITIONS RESULTS 1st : 3 sealed jars

: 3 open jars

2nd: 3 jars covered w/ fine net

Evidence Pro and Con




John Needham put boiled nutrient broth into covered flasks.

CONDITIONS RESULTS

Nutrient broth heated, cooledthen placed in sealed flask



Lazzaro Spallanzani

Showed that fluids heated in sealed flasks did

not contain microbes




Rudolf Virchow / Theodor Schwann /

Matthias Schleiden Cell theory

Louis Pasteur

Is there a “life force” present in air that cancause microbes to develop by spontaneous generation?

Is there a means of allowing air to enter a container but not the bacteria that are present in it?



Conditions Results

Nutrient broth placed in longnecked-flasks, heated, then

sealed

CONCLUSION:



Pasteur’s Work

Microbes are responsible for fermentation and

spoilage of food.Vinegar is produced when bacteria ferments

ethanol in wine.

Spoilage bacteria could be killed heat that wasnot hot enough to evaporate alcohol in wine.

Historical Development :THE GOLDEN AGE OF MICROBIOLOGY



1835: Agostino Bassi discovered a silkwormdisease caused by a ________ .

1865: ________ discovered another silkworm

disease caused by a protozoan.

1840s: Ignaz Semmelweis advocated

_____________ to prevent spread of puerperal

fever.

1860s: ____________ used phenol to prevent

surgical wound infections.

The Germ Theory of Disease



1876: Robert Koch provided experimental

steps (__________) used to prove that a

specific microbe causes a specific disease .

Microbial Etiology of Important diseasesestablished Koch :

•Vibrio cholerae

•Mycobacterium tuberculosis •Bacillus anthracis



Koch’s Postulates



Exceptions to Koch’s Postulates

1. Many healthy people carry pathogens but do notexhibit symptoms of the disease. These“carriers” may transmit the pathogens to otherswho then may become diseased (hospital-

acquired infections, typhoid fever, diphtheria ) .2. Some microbes are very difficult or impossible to

grow in vitro in artificial media (viruses,rickettsias, chlamydias, M. leprae , T. pallidum ) .



3. To induce a disease from a pure culture, theexperimental animal must be susceptible to thatpathogen. Many animals are very resistant to

microbial infections. Many pathogens arespecies-specific. Use of human volunteers aredifficult to find and ethical considerations limittheir use.

4. Certain diseases develop only when anopportunistic pathogen invades a weakenedhost. These opportunists cause disease in a

person who is ill or recovering from another disease. (pneumonia, ear infections following influenza )



Vaccination

1796: Edward Jenner inoculated a

person with cowpox virus resulting to

protection from smallpox.



The Birth of ModernChemotherapy Chemotherapeutic agents used to treat

infectious disease can be synthetic drugs or

antibiotics.

_________ are chemicals produced by bacteria

and fungi that inhibit or kill other microbes.

1910: Paul Ehrlich developed a synthetic arsenic

drug, __________, to treat syphilis.

1930s: Sulfonamides were synthesized.

1928: Alexander Fleming discovered the 1st

antibiotic, ___________ .



HISTORICAL DEVELOPMENT:

Modern Developments in Microbiology

Bacteriology

Mycology

Parasitology

Virology

Genomics, the study of an organism’s genes,

have provided new tools for classifying

microorganisms.



___________ is the study of immunity.

Who proposed the use of immunology toidentify some bacteria according to

serotypes?__________________

Vaccines and IFs are being investigated to

prevent and cure viral diseases.



Paul Berg (1960s) introduced ____________

by inserting animal DNA into bacterial DNA

resulting in the production of an animal

protein.

Recombinant DNA technology or genetic

engineering involves microbial genetics and

molecular biology.



Using microbes

George Beadle and Edward Tatum (1942)

showed that genes encode a cell’s enzymes

Oswald Avery, Colin MacLeod, and Maclyn

McCarty (1944) showed that DNA was thehereditary material.

Francois Jacob and Jacques Monod (1961)

discovered the role of mRNA in proteinsynthesis



Selected Novel Prizes in Physiology orMedicine:

1901 von Behring Diphtheria antitoxin

1902 Ross Malaria transmission

1905 Koch TB bacterium

1908 Metchnikoff Phagocytes1945 Fleming, Chain, Florey Penicillin

1952 Waksman Streptomycin

1969 Delbrück, Hershey, Luria Viral replication1987 Tonegawa Antibody genetics

1997 Prusiner Prions






Microbes and Human Welfare

Modern Biotechnology

Genetic engineering - a biotechnologic

technique . Bacteria and fungi can produce a

variety of proteins (vaccines, enzymes).

____________ : replacement of

missing/defective genes. Genetically modified bacteria - used to protect

crops from insects and freezing.




Flora, microflora = microbes were once classified

as plants

______________ = new term for microbes

Normal Microbiota

They prevent growth of pathogens.

They produce growth factors.

Resistance



Invasive group A Streptococcus Rapidly growing bacteria

cause extensive tissue damage.

Anthrax Bacillus anthracis

Veterinarians and agricultural workers (at risk).

Escherichia coli O157:H7 Enterotoxigenic E. coli

Leading cause of diarrhea worldwide.

BACTERIAL

Emerging Infectious Diseases :



Superbugs:

CRKP

pneumonia, meningitis, UTI, wound infectionsand blood infections

MRSA boils and abscesses resembling infected bug

bites, but can also present as pneumonia or

flu-like symptoms.



West Nile encephalitis West Nile Virus

Bovine Spongiform Encephalopathy

Prions Also causes Creutzfeldt-Jakob disease

Ebola hemorrhagic fever

Ebola virus fever, hemorrhaging, and blood clotting

VIRAL





Bacterial Structure,Classification and GrowthRequirements



Part I.Bacterial StructuresLEARNING OBJECTIVES

• Classify microorganisms according to their morphology and distinct characteristics

• Differentiate between prokaryote and

eukaryote

• Identify the different structures and

functions of a bacterial cell

• Describe bacterial morphology

• Point out ways of classifying bacteria



DIVISIONS OF MICROBIOLOGY

1.VIROLOGY

:Viruses smallest intact infectious

agents

intracellular reproduction only

consist of:

RNA or DNA core

Protein coat

glycoprotein envelope



DIVISIONS OF MICROBIOLOGY

2. PARASITOLOGY

______________:

• Multicellular parasites/worms

PROTOZOOLOGY : ___________

• Unicellular eukaryotic

organisms



3. MYCOLOGY : Fungi

• 2 Forms: _______, _______

• thick cell wall

• Develop from spores or fragments of hyphae

4. PHYCOLOGY : ________

• Mainly aquatic

• contain chlorophyll

• Some produce neurotoxins which can

concentrate in fish / shellfish and cause

poisoning in humans



5. BACTERIOLOGY : Bacteria

• Unicellular, Prokaryotic

• Free living

• Contain both RNA and DNA

• Multiply by _____________

• Eubacteria ; Archaebacteria



• Prokaryotic• Peptidoglycan cell

walls

• characterized byshape, motility &metabolism

B A C T E R I A A R C H A E A

Figure 1.1a

• Prokaryotic• Lack peptidoglycan

• Live in extreme

environments



COCCI (Spherical)

Diplococci

Staphylococci

Streptococci

Tetrad

Octad

BACTERIAL MORPHOLOGY

• SIZE: 0.2 – 2.0 µ m by 1-10 µ m• SHAPE: spherical, rod-shaped, spiral

• ARRANGEMENT



BACILLI (Rod-shaped)

Singles

Diplobacilli

Streptobacilli

Coccobacilli

Palisade SPIRALS

SpirillaSpirochete

Comparison Between Prokaryotic and Eukaryotic CellsComparison Between Prokaryotic and Eukaryotic Cells



Characteristic Prokaryotes Eukaryotes

Size of cell Typically 0.2-2.0 m in diameter Typically 10-100 m in diameter

Nucleus No nuclear membrane or nucleoli

(nucleoid)

True nucleus, consisting of

nuclear membrane & nucleoli

Membrane-enclosed organelles Absent Present; examples include

lysosomes, Golgi complex,

endoplasmic reticulum,

mitochondria & chloroplasts

Flagella Consist of two protein building

blocks

Complex; consist of multiple

microtubules

Glycocalyx /Capsule Present as a capsule or slimelayer

Present in some cells that lack acell wall

Cell wall Usually present; chemicallycomplex (typical bacterial cellwall includes peptidoglycan)

When present, chemicallysimple

Plasma membrane No carbohydrates and generally

lacks sterols

Sterols and carbohydrates that

serve as receptors present

Cytoplasm No cytosketeton or cytoplasmic

streaming

Cytoskeleton; cytoplasmic

streaming

Ribosomes Smaller size (70S) Larger size (80S); smaller size

(70S) in organelles

Chromosome (DNA) arrangement Single circular chromosome;lacks histones

Multiple linear chromosomeswith histones

Sexual reproduction No meiosis; transfer of DNAfragments only (conjugation)

Involves meiosis

Characteristic Prokaryotes Eukaryotes

Size of cell Typically 0.2-2.0 m in diameter Typically 10-100 m in diameter

Nucleus No nuclear membrane or nucleoli

(nucleoid)

True nucleus, consisting of

nuclear membrane & nucleoli

Membrane-enclosed organelles Absent Present; examples include

lysosomes, Golgi complex,

endoplasmic reticulum,

mitochondria & chloroplasts

Flagella Consist of two protein building

blocks

Complex; consist of multiple

microtubules

Glycocalyx /Capsule Present as a capsule or slimelayer

Present in some cells that lack acell wall

Cell wall Usually present; chemicallycomplex (typical bacterial cellwall includes peptidoglycan)

When present, chemicallysimple

Plasma membrane No carbohydrates and generally

lacks sterols

Sterols and carbohydrates that

serve as receptors present

Cytoplasm No cytosketeton or cytoplasmic

streaming

Cytoskeleton; cytoplasmic

streaming

Ribosomes Smaller size (70S) Larger size (80S); smaller size

(70S) in organelles

Chromosome (DNA) arrangement Single circular chromosome;lacks histones

Multiple linear chromosomeswith histones

Sexual reproduction No meiosis; transfer of DNAfragments only (conjugation)

Involves meiosis



General Structure

of a Prokaryote



A. Flagella

PARTS

filament Hook

basal body

Flagella rotates to move Flagellin (H Ags)

Purpose: ___________________

Flagellar Arrangement



Flagellar Arrangement

Figure 4.7

1. Monotrichous : _______

flagellum at one end

2. ____________ : small

bunches arising from one

end of cell3. Amphitrichous:

____________________

4. ____________ : flagella

dispersed over surface of

cell



Internal Flagella

• Also known as: ___________________

• Periplasmic filaments

• enclosed between cell wall & cell

membrane of spirochetes

• motility

B A d f A h



B. Appendages for Attachment

• fine hairlike bristles from the

cell surface

• function in adhesion to othercells and surfaces

FIMBRIAE

Pili



Pili

• Appendages for Mating rigidtubular structure

•Made up of: __________

• in ____________ cells only

Functions:

joins bacterial cells for DNAtransfer

adhesion

C B i l S f C i



C. Bacterial Surface Coating

• external to the cell wall

• Made of sugars and/or proteins

FUNCTIONS

________________

________________

________________

Glycocalyx



Glycocalyx

2 TYPES:

1.capsule _____________________

2.slime layer _____________________



The Cell Envelope: Cell Wall

• peptidoglycan

• provides strong, flexible support to the

bacterial cell

• Maintains cell integrity

• N-acetylglucosamine (NAG)

• N-acetylmuramic acid (NAM)• Linked by ____________



Figure 4.13a



4 Groups Based on Cell Wall

Composition1. Gram positive cells

2. Gram negative cells3. Bacteria without cell walls

4. Bacteria with chemically unique cell walls



Gram Positive Cell Wall

Consists of :

thick peptidoglycan

tightly bound acidic polysaccharides

cell membrane

Retain crystal violet and stain ________



Gram Negative Cell Wall

Consists of: outer membrane with lipopolysaccharide

thin shell of peptidoglycan

periplasmic space

inner membrane LPS

endotoxin

may function as

receptors and blocking

immune response

contains ________ proteins in upper layer

Lose crystal violet and

stain _______ from

_________ .Protective structure while

providing some flexibility

and sensitivity to lysis





The Gram Stain

• Differential stainGram-negative

Gram-positive

• Important basis of bacterial classification andidentification

• Practical aid in diagnosing infection and

guiding drug treatment





Atypical Cell Walls

• Some bacteria lack typical cell wall structure Mycobacterium and Nocardia

Gram-positive cell wall structure with lipid ____________.

• basis for ____________________

• Some have no cell wall

Mycoplasma

cell wall is stabilized by ___________



Cytoplasm

• dense gelatinous solution of

sugars, amino acids, & salts

• 70-80% water

• serves as solvent for materialsused in all cell functions

Ch Pl id



Chromosome

• single, circular,double-stranded

DNA molecule

• contains all the

genetic informationrequired by a cell

• DNA is tightly coiled

around a protein

Plasmids

• Self-replicating,small circles of DNA

• may encode

antibiotic

resistance,tolerance to toxic

metals, enzymes &

toxins

Inclusions &



• intracellular storagebodies

Examples: Glycogen

gas vesicles

carboxysomesPolyphosphate

granules

Inclusions &Granules

Ribosomes

• prokaryotic differ from

eukaryotic ribosomes

in size & number of

proteins• site of ________

synthesis

• all cells haveribosomes

Endospores



Endospores

• Resting cells

• Resistant to heat, radiation

& chemicals

• Examples: _________,

_________

___________:

Endospore formation ___________:

Return to vegetative state



Endospores

• resistance linked to high levels

of calcium & some acids

• longevity verges on immortality

• Control: pressurized steam at120oC for 20-30 minutes

Bacterial Morphology



Bacterial Morphology



Part II



Part II.Classification of Bacteria

Learning outcomes

Students should be able to:

• Understand the basic principles of microbial classification systems.

• Be familiar with structural and biological

characteristics to classify bacteria

Interrelated areas of taxonomy:



y

• Classification

– the arrangement of organisms into taxonomic groupson the basis of similarities or relationships.

• ________________

- naming an organism by international rules according to

its characteristics.

• Identification

(1) to isolate and distinguish desirable organisms from

undesirable ones;(2) To verify the authenticity or special properties of a

culture, or in a clinical setting;

(3) To isolate and identify the causative agent of a disease

Classification Systems



y

Numerical Taxonomy

• the computer clusters different strains based onthe frequency with which they share traits.

Phylogenetic Classification System

• Groups reflect genetic similarity andevolutionary relatedness

Phenetic/Phenotypic Classification System

• Groups are based on convenient, observable

characteristics.





Useful Properties in Classification

•Colony morphology

•Cell shape & arrangement

•Cell wall structure (Gram staining)

•Special cellular structures

•Biochemical characteristics



Levels of Classification

• Kingdom (not used by most bacteriologists)

• Phylum/Division

• Class

• Order

• Family

• Genus (plural: Genera)

• Species (both singular & plural)



Species:

Classic definition : A collection of microbialstrains that share many properties and differ

significantly from other groups of strains.

Species are identified by comparison with

known “type strains” -- well-characterized

pure cultures - references for the identification

of unknowns.



Strain:

• A population of microbes descended from a

single individual or pure culture.

• Different strains represent genetic variability

within a species.

_________ : Strains that differ in biochemical or

physiological differences.

_________ : Strains that vary in morphology.

_________ : Strains that vary in their antigenic

properties

Nomenclature



Nomenclature

Scientific name (Systematic Name)

• Species name is never abbreviated.

• A genus name may be used alone to indicate

a genus group.• A species name is never used alone.

• Common or descriptive names (trivial names)

Types of Diversity



yp y

• Metabolic diversity

• Structural diversity

• Morphological diversity

• Genetic diversity

Bergey’s Manual of Systematic Bacteriology



Bergey’s Manual of Systematic Bacteriology

• main resource for determining the identity

of bacteria species, utilizing every characterizingaspect.

• Use successive "key" features to narrow downidentification

• Primary emphasis is phylogenetic, not phenetic

Dichotomous Key



Dichotomous Key



Part III



Part III.Physical and Nutritional Growth

Requirements of Bacteria

• Identify the growth requirements of bacteria

• Appreciate the importance of the

physiological and nutritional requirements for bacterial growth



PHYSIOLOGY

study of vital life processes of organisms

& how these processes normally function inliving organisms

NUTRITIONAL REQUIREMENTS substances required for energy generation

and cellular biosynthesis.

chemicals and elements of theenvironment that are utilized for bacterialgrowth

Table 1. Major elements, their sources and functions in bacterial cells.



j ,

Element% ofdryweight

Source Function

Carbon 50organic compounds orCO2

Main constituent of cellular material

Oxygen 20H2O, organiccompounds, CO2, and O2

Constituent of cell material and cell water; O2 is electron acceptor inaerobic respiration

Nitrogen 14NH3, NO3, organiccompounds, N

2

Constituent of amino acids, nucleic acids nucleotides, and coenzymes

Hydrogen 8H2O, organiccompounds, H2

Main constituent of organic compounds and cell water

Phosphorus 3inorganic phosphates(PO4)

Constituent of nucleic acids, nucleotides, phospholipids, LPS, teichoicacids

Sulfur 1SO4, H2S, So, organicsulfur compounds

Constituent of cysteine, methionine, glutathione, several coenzymes

Potassium 1 Potassium salts Main cellular inorganic cation and cofactor for certain enzymes

Magnesium 0.5 Magnesium salts Inorganic cellular cation, cofactor for certain enzymatic reactions

Calcium 0.5 Calcium saltsInorganic cellular cation, cofactor for certain enzymes and a component ofendospores

Iron 0.2 Iron saltsComponent of cytochromes and certain nonheme iron-proteins and acofactor for some enzymatic reactions

TYPES OF ORGANISMS BASED ON PHYSIOLOGIC



REQUIREMENTS:

Nutritional type Energy sourcePhototroph Chemotroph

a.Chemolithotrophs

b.Chemoorganotrophs

Inorganicchemicals

Organicchemicals

Autotroph

Heterotroph

Metabolic Diversity Among Organisms



Nutritionaltype

Energysource

Carbonsource Example

Photoauto-troph Light CO2 Oxygenic: ______________

Anoxygenic: ______________

Photohetero-

troph

Light Green, purple

nonsulfur bacteria.Chemoauto-troph

Chemical CO2 Iron-oxidizing, sulfur,hydrogen, nitrifyingbacteria.

Chemohetero-

troph

Chemical Most bacteria,

fermentative bacteria,animals, protozoa,fungi.

Growth Factors



Growth Factors

• are essential substances that the organism is

unable to synthesize

• required in small amounts for biosynthesis

CATEGORIES:

Purines and pyrimidines

Amino acids

Vitamins





Group Superoxidedismutase

Catalase Peroxidase

Obligateaerobes &

most

facultative

anaerobes

Most

aerotolerant

anaerobesObligate

anaerobes

@ Don’t forget to fill in the blanks!

THERMAL REQUIREMENT



THERMAL REQUIREMENT

PSYCHROPHILES

prefer cold temperatures

cause food spoilage

_________________

MESOPHILES

prefer moderate temperature

THERMOPHILES prefer high temperatures

________________



• the solvent in which the molecules of life aredissolved

• Supply depends on: relative humidity and wateractivity (A

w

).

• Aw = affected by the presence of solutes thatare dissolved in the water.

• The higher the solute concentration of asubstance, the lower is the Aw and vice-versa.



• SALT: only common solute in nature that occurs

over a wide concentration range

• ____________: microorganisms that require some

NaCl for growth.

• Mild halophiles

• Moderate halophiles

• Extreme halophiles

• _____________ = grows at moderate saltconcentrations, even though they grow best in the

absence of NaCl.

• Xerophiles = organisms which live in ___________ .



Movement across membranes:

_________diffusion :

• Movement of a solute from an areaof high concentration to an area of

low concentration _________ diffusion :

• Solute combines with transporter protein in membrane

Movement Across Membranes



Osmosis – Mov’t of H2O across a selectively

permeable membrane from an area of

↑ H2O conc’n to an area of ↓ H2O.

Osmotic pressure

– pressure req’d. to stop H2O mov’tacross the membrane.

Figure 4.18a

pH REQUIREMENT



pH REQUIREMENT

the acidity or alkalinity of a solution.

• ACIDOPHILE• NEUTROPHILE

• ALKALIPHILE

Nutritional and Physical



yRequirements for Bacteria Growth.

• Major and trace elements

• Carbon and energy sources

• Growth factors• Oxygen, Carbon dioxide

• Temperature

• Water / Moisture

• pH requirement



Bacterial Growth,

Genetics

and

Control of Bacterial Growth

Part I: Bacterial Growth



Part I: Bacterial Growth

• Describe the different phases of thebacterial growth curve

• Choose the appropriate culture media for bacterial growth

• Synthesize the importance of the

processes involved in culturing bacteria

Bacterial Growth



Bacterial Growth

Growth = an orderly increase in the quantityof cellular constituents.

____________ = asexual reproduction.

increase in cell mass & number of ribosomes

duplication of bacterial chromosome

cell wall & plasma membrane synthesis

chromosome partitioning & septum formation

cell division.

• Binary or transverse fission



Generation Time



If a single bacterium reproduce every 20minutes, how many would there be in 2 hrs?

the time it takes for an organism to

double its number time required for a cell to divide

If S. aureus has a generation time of 60minutes, how many cells would be in 7 hrs?

Bacterial Growth Curve



A. Lag phase



g p

Organism absorbs nutrients, synthesizes

enzymes & prepares for reproductionB. __________

cellular reproductive stage; number doubles

with each generation time

C. Plateau phase organisms maintain their greatest population

density

D. ___________ rapid decline in cells until there is complete

cessation of reproduction.



• Nutrients for microbial growth

• Inoculum: Microbes introduced into medium

• Culture: Microbes growing in/on a medium

• ___________: Contains only one

species or strain

• _________: A population of cells arising

from a single cell or spore.

Types of Culture Media



According to Consistency:

Liquid media

Semi-solid

Solid media

According to Composition:

Synthetic medium Non-synthetic medium

According to Function/Purpose :



g p

General purpose

Enriched Selective

Differential

Anaerobic growth

Specimen transport

Assay

Enumeration

Anaerobic Culture Methods



Figure 6.5

Types of Colonies



Types of Colonies

• Mucoid colony• Smooth colony

• Rough colony

Measurement of Cell Numbers



1. Direct microscopic counts Dead cells cannot be distinguished from livingones. Samples must be concentrated bycentrifugation or filtration to increase sensitivity.

2. Electronic counting chambers Count numbers and measure size distributionof cells.

3. Indirect viable cell countsspreading a sample of culture on a nutrient agar surface. On a suitable medium, each viable unitgrows and forms a colony.

Part II: BACTERIAL GENETICS



Part II: BACTERIAL GENETICS

Each student will be able to:

• Describe the characteristics of a bacterial

genome;

• Identify mechanisms of gene transfer;

• Recognize the great clinical importance of the

ability of bacteria to transfer genes, especially

genes for antibiotic resistance, to other bacteriaboth within and between species.

The Bacterial Genome



The Chromosome

• single, long piece of circular, double-

stranded DNA

• Carry all of the essential genes and many

nonessential genes of the bacterium• Contain 2000 to 4000 genes

The Bacterial Genome



Plasmids

• Small DNA circles

• Replicate independently (1 or more)• Genes for toxins, proteins that promote

transfer

GENETIC TRANSFER



• ____________ Gene Transfer

– When organisms replicate their genomes and

provide copies to descendants

• ____________ Gene Transfer – Acquisition of genes from other microbes of

the same generation,

– can be a different species, or even a different

genus than the donor.

HORIZONTAL GENE TRANSFER



HORIZONTAL GENE TRANSFER

1) Conjugation

2) Transformation

3) Transduction

CONJUGATION







TRANSFORMATION



TRANSDUCTION



BACTERIOPHAGE (phage)



BACTERIOPHAGE (phage)

• a virus that replicatesinside a bacterial cell

• Consists of a nucleic

acid encapsulated in aprotective protein coat

• DNA or RNA,

single/double stranded,3000-200,000 bases

Types of Phages



• Virulent phage(T-phages)

• Temperate phage

(phage lambda)



LYSOGENIC CONVERSION



LYSOGENY

Cells are immune to reinfxn

by same phage.

Cells may exhibit newproperties.

Allows phage to take a bit

of the adjacent bacterialDNA

GENETIC VARIATION



A. _____________

any change in the DNA base sequence

B. MOBILE GENETIC ELEMENTS

______________ = DNA segments that have

the ability to move from place to place on the

chromosome and into and out of plasmids

Probably responsible for most of the genetic

variability & the spread of antibiotic resistance

genes

Part IIIC t l f Mi bi l G th



Control of Microbial Growth

• Discuss concepts, principles &significance of infection control &laboratory safety

• Discuss various of sterilization &disinfection methods with emphasis on

temperature, time, principles/mechanism involved, advantages &disadvantages

TERMINOLOGIES:



Sterilization: Killing or removing

________________ in a material or an object.

Commercial Sterilization: Heat treatmentthat kills endospores of _______________, the

causative agent of botulism.

_____________: Reducing the number of pathogenic microbes to the point where they no

longer cause diseases.



Disinfectant: Applied to ______________

Antiseptic: Applied to ______________

Degerming: Mechanical removal of most

microbes in a limited area.

_________ : Use of chemical agent on food-

handling equipment to meet publichealth standards and minimize

chances of disease

transmission.

Sepsis : Indicates bacterial contamination.



________ : Absence of significant contamination.

Aseptic techniques : prevent contamination of

surgical instruments, medical personnel,

and the patient during surgery.

Bacteriostatic Agent : An agent that ________

the growth of bacteria, but does not

necessarily kill them.Germicide : An agent that kills certain microbes.



Physical Methods of Microbial Control:



A. HEAT : Kills microorganisms by _____________ ________________________________.

Thermal Death Point: Lowest T° at which all

of the microbes in a liquid suspension will be

killed in 10 minutes.

Thermal Death Time: Minimal length of time in

which all bacteria will be killed at a given T° .

Decimal Reduction Time: Time (mins.) at

which 90% of bacteria at a given T° will be killed.

A.1. Moist Heat:



BOILING :

Heat to 100oC or more.

Kills vegetative forms of bacteria, most viruses,

fungi (and spores) within 10 mins or less.

Endospores & some viruses are not easily

destroyed.

AUTOCLAVE : Chamber which is filled with hot

steam under pressure



steam under pressure.

steam T° reaches ___ oC at ___ psi for ___ minutes.

PASTEURIZATION: reduces microbesresponsible for spoilage of beverages.

Classic Method of Pasteurization

High T ° Short Time Pasteurization Ultra High T ° Pasteurization

A.2. Dry Heat: Kills by ____________ effects.



Direct Flaming

Incineration

Hot Air Sterilization

B. Filtration: Removal of microbes by passage of a liquid or gas through a screen-likematerial with small pores.

High Efficiency Particulate Air Filters (HEPA)

Membrane Filters

C. Low Temperature



REFRIGERATION:

0 to 7oC.

Reduces metabolic rate of most microbes so

they cannot reproduce or produce toxins.

FREEZING: below 0oC.

Flash Freezing

Slow Freezing

D. ___________: without water, microbes cannotgrow or reproduce, but some may



grow or reproduce, but some mayremain viable for years.

E. Osmotic Pressure : increased concentrationsof salt & sugar in substances canincrease osmotic pressure & createa ____________ environment.

________________ :

As water leaves the cell, plasma membrane shrinksaway from cell wall. Cell may not die, but usuallystops growing.

F. Radiation:

________________ Radiation:



• Dislodge electrons from atoms and form ions.• Cause mutations in DNA and produce peroxides.

• Sterilize pharmaceuticals & disposable medical supplies.

________________ Radiation:

• Damages DNA by producing thymine dimers,

which cause mutations.

• Used to disinfect ORs, nurseries, cafeterias.

Microwave Radiation:



Heat is absorbed by water molecules.

May kill vegetative cells in moist foods.

Bacterial endospores are not damaged by

microwave radiation.

Solid foods are unevenly penetrated bymicrowaves.

Trichinosis outbreaks have been associated with

pork cooked in microwaves.



B. HALOGENS: Effective alone or in compounds.



Iodine:

Tincture of iodine Iodophors

Chlorine: When mixed in water forms ________________ :

Cl2 + H2O ------> H+ + Cl- + HOCl

to disinfect drinking water, pools, and sewage.

Chlorine is easily inactivated by organicmaterials.

C ALCOHOLS



C. ALCOHOLS:

Kill bacteria, fungi, but not endospores or

naked viruses.

Used to mechanically wipe microbes off

skin before injections or blood drawing.

Not good for open wounds, becausecause proteins to coagulate.

D. HEAVY METALS:

Oli d i ti



Oligodynamic action

Silver: 1% AgNO3

Mercury:

merthiolate , mercurochromeCopper

Copper sulfate

SeleniumZinc

Zinc chloride, Zinc oxide

E. QUATERNARY NH4 COMPOUNDS (Quats) :



surface active agents; cationic detergents.

Effective against GPB, less effective against GNBbacteria.

Also destroy fungi, amoebas, and envelopedviruses.

Advantages: Strong antimicrobial action, colorless,odorless, stable, and nontoxic.

Diasadvantages: Organic matter interferes witheffectiveness. Neutralized bysoaps & anionic detergents.

F. ALDEHYDES:



Formaldehyde gas

Excellent disinfectant. ____________ (37% aqueous solution)

Irritates mucous membranes, strong odor.

Glutaraldehyde Less irritating, more effective than formaldehyde.

sterilizing agent.

Commonly used to disinfect hospital instruments.

G. Gaseous Sterilizers:



Denature proteins, by replacing functionalgroups with alkyl groups.

Ethylene Oxide:

Kills all microbes & endospores

Exposure Time : ____________.

Toxic and explosive in pure form.

H. Peroxygens (Oxidizing Agents): Oxidize cellular components of treated microbes



Oxidize cellular components of treated microbes.

Disrupt membranes and proteins.

Ozone:

• Highly reactive form of ________

• Used along with chlorine to disinfect water.

• More effective killing agent than chlorine, but

less stable and more expensive.

• Made by exposing oxygen to __________ or

____________.

Hydrogen Peroxide:

Used as an antiseptic



Used as an antiseptic.

Effective in disinfection of inanimate objects. Sporicidal at higher temperatures.

Benzoyl Peroxide:

Used in acne medications.

Peracetic Acid:

effective liquid sporicide

Sterilant

does not leave toxic residues.



Figure 7.11



SPECIMEN COLLECTION,

PROCESSING

& HANDLING

LEARNING OBJECTIVES:



Each student is able to:• Describe the general principles for specimen

handling;

• Select the appropriate specimen collection,transport and processing method;

• Relate the significance of specimen collection

and handling in the pre-analytical steps in theaccurate diagnosis of infectious diseases.

Specimen Collection:General Guidelines



Obtain specimen before treatment

Collect material from the appropriate site

Collect specimen during acute stage of illness

Collect specimen properly & asepticallyCollect sufficient quantity

Label all specimen

Transport the specimen immediatelySpecimen should be accompanied with arequest form

MAJOR APPROACHES TOAVOID CONTAMINATION



AVOID CONTAMINATION

• Disinfectant the area

• Bypass area with normal flora

• Culture for specific pathogen only

• Quantitate by colony counting

• Sufficient quantity of specimen

• Prompt delivery to the laboratory

Successful Recovery ofEti l i A t D d



Etiologic Agents Depends on:

Advanced planning

Collection of appropriate and

adequate specimenCorrect packaging and rapid transportto the laboratory

Ability of the laboratory to accuratelyperform the diagnostic tests

CLINICAL SPECIMEN



• Blood & blood components• Respiratory

• Urine

• Cerebrospinal fluid• Stool / Rectal swab

• Exudates / Transudates

Critical Delivery Time for BiologicalSpecimen



Specimen Delivery time

Respiratory 1 hour

Gastrointestinal 1 hour

Blood culture 1 hour

Anaerobic 30 mins

CSF Immediately

Other fluids Immediately





Urine 1 hr

Wound, skin & soft tissue 30 mins

Fungal 1 hr

Mycobacterial 1 hr

Chlamydia 1 hr





BLOODGENERAL CONSIDERATIONS:



GENERAL CONSIDERATIONS:

Collect blood during febrile episode

During a chill

A (+) Blood culture depends on thepathogenic process of the organism.

Obtain 2-3 simultaneous blood cultures from

different sites per 24 hours. A single (-) bloodculture does not rule out bacteremia.

Collect blood aseptically.

VOLUME :



Young Children: ______in ____ml broth

Adults: __________in 50ml broth

If Px is on penicillin, administerpenicillinase

Not effective against methicillin, cloxacillin,

nafcillin

Hold for 2 weeks before reporting as (-)

BLOOD CULTURES CAN BE OBTAINED IN

THE FOLLOWING CONDITIONS :



THE FOLLOWING CONDITIONS :

In acute illnesses

Fever of unknown origin

In cases of patients with acuteinfective endocarditis

In cases of suspected bacterial

endocarditis



Turbidity

Hemolysis

Colony or pellicle formation

Presence of gas or bubble

MEDIA USED:

T ti S B th C l bi B th



Trypticase Soy Broth Columbia Broth

Brain –Heart Infusion Brucella Broth

Castaneda medium Thioglycollate Broth

ANTICOAGULANT:

Sodium Polyanethol Sulfonate

Neutralizes bactericidal effect of serum

Prevents phagocytosis

Inactivates some antimicrobial agents

COMPONENT OF BLOOD CULTURE BROTH

1 % Gelatin



enhance growth of Neisseria meningitidis 0.1 % Bacto-agar enhance growth of anaerobic organism

anticoagulant

0.025 % Sodium Polyanetholsulfonate

inhibit activity of complement & lysosyme

prevents phagocytosis inactivates therapeutic concentration of

aminoglycosides

Blood Culture Technique



Find the puncture site, if arm vein

is selected, apply a tourniquet

Clean the skin with iodine, followed

by 70% alcoholClean the rubber stopper with

alcohol swab

Collect the required amount ofblood

PROCESSING OF BLOOD CULTURES

I l bl d i b h i i f 1 10



Inoculate blood in broth in a ratio of 1:10

Incubation Temperature: 350C - 370C

Incubation Time: 7 days

Subculture on BAP & CAP: after 14-17 hrs,

3 days, 5 days, 7 days

BLOOD COMPONENTS



• Organisms found in fatal transfusionreactions are psychrophilic.

• Gram (-) bacilli

• PLATELET



Nasopharyngeal swab

Throat swab

POSSIBLE PATHOGENS OF THE URT :S. pneumoniae, S. pyogenes S.aureus C. diphtheriae Haemophilus influenzae Klebsiella spp.Other Enterobacteriacae

Throat Swabbing



MEDIA for URT specimen:

H. influenzae :CAP or BAP w/ Staph

N. meningitidis :CAP/Thayer Martin

B. pertussis :Charcoal Cephalexin

Culture must include anaerobicconditions for β streptococcus.

B L R i t T t



B. Lower Respiratory Tract

Sputum

Tracheal aspirate

Broncho alveolar Lavage

Bronchoscopy secretions

S. pneumoniae

S. aureus

H. influenzae Moraxella catarrhalis

Legionella spp .

Mycobacterium spp.

Mycoplasma spp.

Klebsiella spp.

Enterobacteriacae

Collection & Transport of Sputum



Use a dry, wide-mouthed bottle

Collect sample early morning

Transport ASAP

may be refrigerated but examinedw/in 2 –3 hrs

PROCEDURE:



PROCEDURE:

Make a direct smear

Do concentration technique

Culture

Sputum Classification Based on WBCs &



Sputum Classification Based on WBCs &Squamous Epithelial Cell Densities

Cell numbers per x 100 (low power) field

Group WBCs Epithelial Cells

6 <25 <55 >25 <104 >25 10-25

3 >25 >252 10-25 >251 <10 >25

Container: Sterile wide mouth glass or



g

plastic jar with tight-fitting lids.Method: Suprapubic aspiration

Cystoscopy or catherization

Clean - catch midstream

Collection: Early morning

Processing: Within 2 hrs after collection

GS of uncentrifuged urine



PROCEDURE:

Inoculate spx in BAP, EMB, Mac using acalibrated loop

Incubate overnight

Observe for 48 hrs before reportingnegative

Actual # ofcolonies

xCalibration

of loop=



POUR PLATE METHOD:

• 1:100 dilution of urine w/ sterileH2O

• incubate for 18 –24 hrs

• colony count x 100 = bacteria / mL

POSSIBLE PATHOGENS



Enterobacteriacae

S. aureus

S. saprophyticus Enterococcus spp.

Yeast

CEREBROSPINAL FLUID



The specimen should be taken beforeantibiotics are administered.

Antigen tests may give a specific

diagnosis even after antibiotic treatment.

CSF should always be taken from

patients with suspected purulent

meningitis, regardless of antibiotic

treatment.

CSF Specimen

Volume :



Volume : ___________________

Collection Time : __________________

1st tube = cell count, differential staining

2nd tube = Gram’s stain & culture

3rd tube = protein, glucose & special tests

PROCEDURE:



Centrifuge CSF;

Make a smear for GS and india ink

Culture CSF on recommended media

MEDIA:Trypticase Soy Broth / thioglycollate

BAP for Gram (+) cocci

CAP for Gram (-) cocci

EMB/Mac for Gram (-) bacilli

• CSF for bacterial culture:Incubate for not > 12 hrs OR;



Stand at room T° not > 1 hour DO NOT REFRIGERATE!

• CSF for viral culture:Refrigerate immediately

If held for more than 24 hrs, freeze

specimen at –700

C

Order of Tests for Scanty Sample:



Order of Tests for Scanty Sample:

1) Culture

2) Bacterial Antigen Detection

3) Gram Stain

4) Cell Count

5) Other clinical pathology tests



MAJOR PATHOGENS



MAJOR PATHOGENS

Streptococcus pneumoniae

Haemophilus influenzae type bNeisseria meningitidis

Ideal specimen:

STOOL



Freshly collected stool on the earlystage of a disease

Rectal swab may be used

Amount: _______________

Container: Clean, wide mouth with lid .

Transport time : 2 hours after collection

Transport medium : _____________________

PROCEDURE:

Put rectal swab in enrichment broth or



u ec a s ab e c e b o o

transport mediumGS is NOT usually done but helps in

identifying etiologic agents

• Gram + cocci in clusters

• Gram – comma-shaped bacilli

• Gram + bacilli in large numbers

• Gram – bacilli

Stool Culture : 1st day



Inoculate the spx and incubate it overnight

MEDIA:

• Differential

• Selective

• Enrichment

Stool Culture : 2nd day



Stool Culture : 2nd day

Check diff’l. media for LFs & NLFs

With growth : Subculture and do

biochemical tests

No growth : inoculate culture from

enrichment media intoEMB or Mac

Stool Culture : 3rd day

Note patterns of biochemical reactions



Note patterns of biochemical reactions

If suggestive of Salmonella, Shigella,Vibrio, DO serological typing

If growth occurs after doing step 3 (2nd

day), DO biochemical test

Incubate overnight and perform step 1 of

day 3

EXUDATES & TRANSUDATES



WoundsBoils

Abscesses

UlcersGranules

Rash

GastricAspirates

TissuesEye discharge

Ear discharge

EffusionsEndocervical

Urethral

Anorectaldischarge

COLLECTION1. Discharge / Fluids : best to aspirate



a. Dry wound – moisten swab w/ NSSbefore collecting

b. Skin lesion – remove crust of pustule/

vesicle cap then gentlyswab lesion

Punch biopsy Tzanck smear

c. Endocervical : use swab

d Urethra : use swab or scrape



d. Urethra : use swab or scrape

mucosa of anterior urethra

e. Anorectal : insert swab about 4-5cm. into the anal canal

Possible Pathogens InAnogenital Specimen:



Anogenital Specimen:

T. pallidum

N. gonorrheae

C. trachomatis

C. albicans

G. vaginalis



2. Irrigation, Intravenous orIntra-arterial Catheters

3. Tissue (scrapings,etc)

EXUDATES / TRANSUDATES:

Aspirates : Sterile vial



Ulcerative lesions : biopsy in sterile vialw/o preservatives

Irrigation, Intravenous

Intra-arterial Catheter tips

Swabs : 2 pc. in a sterile tube

Fluids : syringe with sterile rubber stopper Corneal scraping : direct inoculation or

scraping of instruments

sterile vial

Transport time : ASAP or max. of 2 hours



Delay in transport :

Refrigerate

If dry swabs, place in TSB or Thioglycollate

Amies or Stuart Transport Medium

SBAP slant

Transport Medium



•

N. gonorrhoeae

Fastidious organisms

stool



Nothing is more important than the

appropriate selection, collection, and

handling of specimen for

microbiologic diagnosis.



METHODS

OFIDENTIFICATION

LEARNING OBJECTIVES:



• Discuss concepts and

principles of the techniques

by which bacteria are

identified in the laboratory.



•MICROSCOPY & STAINING•CULTURE METHODS

•

BIOCHEMICAL TESTS•SEROLOGICAL TESTS

•ANIMAL INOCULATION

I. MICROSCOPIC METHOD



Microbes can be viewed in the ff. states:

A. LIVING STATE

• Visualize size, shape & arrangement• Check motility

B. FIXED STATE

METHODS OF FIXATION:



METHODS OF FIXATION:

HEAT FIXATION

METHANOL FIXATION

PURPOSE:

•Kills & preserves the organism

•

Anchors the smear to the slide

STAINING

• process of adding stains or dyes for



• process of adding stains or dyes for

enhanced visualization of the microbe

MICROBIAL STAINING METHODS:

SIMPLE

SPECIAL

DIFFERENTIAL

A Simple Stains

Types of Stains



A. Simple Stains

Use of a single basic dye

B. Special Stains

Heat is required to stain endospores.

Flagellar stains need mordants to enlarge

flagella.

SPECIAL / STRUCTURAL STAIN

Capsule Welch, Anthony’s, Gin’s, Hiss,



Tyler’s, Muir’s, India ink Cell wall

Metachromatic Granules

Albert’s, Ljubinsky, LAMB,

Neisser’s

Flagella

Endospore Dorner’s, Schaeffer & Fulton/Wirtz-Conklin, Glacial acetic acid



GENERAL RULES:

• All cocci are gram (+) EXCEPT



g

Neisseria, Branhamella/Moraxella, &Veilonella.

• All bacilli are gram (-) EXCEPT

Mycobacterium, Corynebacterium,Lactobacillus, Listeria, Clostridium,Bacillus, Erysipelothrix, and Nocardia.

• All spiral bacteria are gram (-) whenstained.

THEORIES OF GRAM STAINING

GRAM (+) GRAM (-)

Mg-RNA MgRNA + CV - I2 MgRNA is



Mg-RNATheory

MgRNA + CV I2

complex = insolublecompd.

MgRNA is

absent

Benian

Theory

Less permeable cell

walls

Permeable cell

walls

Stearn &Stearn

Theory

isoelectric ptmaking them acidic;

bind well w/ basicdye

isoelectric ptmaking them

basic; bind wellw/ acidic dye

Lipidcontent

lipid content but teichoic acid

lipid content;no teichoic acid



Non –Stain System to Determine TrueGram Stain Reaction



• L – alanine, 4 – nitroanilite (LANA) • Turns yellow_________ when

touched to a colony of gram

negative bacteria

• 3% KOH

• Formation of string –like materialindicates _____________organisms

ACID FAST STAINING

• MYCOLIC ACID – responsible for the acid



fastness of mycobacteria

GENERAL RULE:

All organisms are non-acid fast EXCEPT:

• Mycobacterium

• Nocardia spp.

• Corynebacterium spp.



Carbolfuchsin Red RedHeat Red Red

Acid-alcohol Red Colorless

MethyleneBlue

Ways To Facilitate AFB staining:



Using steam

Increase concentration of phenol &

basic fuchsin

Prolonged contact time

Adding wetting agent

TYPES OF ACID FAST STAINING

1. ZIEHL – NEELSEN METHOD



uses Heat (hot stain)2. KINYOUN METHOD

Uses wetting agents

3. PAPPENHEIM’S

Differentiates M. tb from M. lacticala and M.smegmatis

4. BAUMGARTEN Differentiates blue M. tb & red M. leprae

RECALL :

II. CULTURE METHOD



• Culture Medium

• Inoculum

• Agar

• Colony

Types: mucoid, smooth, rough

• Culture Types: pure, mixed, stock

METHODS OF OBTAINING PURE CULTURE



B. POUR PLATE

C. SELECTIVE METHOD

D. ANIMAL INOCULATION

A. STREAK PLATE

TYPES OF MEDIARECALL:



1. Physical form (consistency)2. Distribution

3. Chemical composition 4. Functional type (use)

Physical Form: LIQUID MEDIA



• with solidifying agent• detects bacterial motility

Physical Form: SEMI – SOLID MEDIA

• water – based• broth, milk, infusion

Physical Form: SOLID MEDIA



LIQUEFIABLE

NON – LIQUEFIABLE

DISTRIBUTION

• TUBED• PLATED – sterile petri dish

Functional Type:GENERAL PURPOSE MEDIA



• Designed to grow a broad spectrum of microbes

• Examples:

– Nutrient agar – Nutrient broth

– Trypticase Soy Agar

Functional Type: ENRICHMENT

Functional Type: ENRICHED



• Similar w/ enrichedexcept that the

media is liquid

• with substances thatallow growth of

fastidious organisms

• blood, serum, Hgb,vitamins, AA

Functional Type: SELECTIVE



• contains agents that inhibit growth of some microbes

• permits preliminary identification of

genus or spp.

Examples:

Mannitol Salt Agar

Hektoen Enteric Agar

MEDIUMSELECTIVE

AGENTUSES



MuellerTellurite Potassiumtellurite Isolation of C.diphtheriae

Enterococcus faecalis broth

Na azide

Tetrazolium

Isolation of fecal

Enterococci

Phenylethanolagar

Phenylethanolchloride

Isolation of Staph & Strep

Tomato juice

agar

Tomato juice,

acid

Isolation of

Lactobacilli

MacConkey Bile, crystal violetIsolation of

Gram (-) enteric

MEDIUMSELECTIVE

AGENTUSES



SSA Bile, citrate,

brilliant green dye

Isolation of Salmonell a &

Shigella

Lowenstein – Jensen agar

Malachite greendye

Isolation &maintenance of

Mycobacteria

Sabouraud’s agar

pH 5.6 Isolation of fungi

Functional Type: DIFFERENTIAL



• displays visible differences amongmicrobes

Examples:

MacConkey agarNeutral red

Spirit blue agar

Spirit blue dye

MEDIUMSubstance for

differentiation

Differentiates

between

BAP Intact RBCsTypes of



BAP Intact RBCshemolysis

MSAMannitol, phenol red,

7.5% NaCl

Species of

Staphylococcus

HEABromthymol blue, acidfuchsin, salicin, citrate,

sucrose, thiosulfate,

ferric ammonium, bile

Salmonella,Shigella , other

LFs & NLFs

SBA Spirit blue dye,oilFat-utilizing

bacteria from those

that do not

MEDIUMSubstance for

differentiation

Differentiates

between

Urea Urea-hydrolyzing



broth Urea, phenol red

y y g

bacteria

SIM Thiosulfate, Fe H2S gas production

TSI Triple sugars, Fe,phenol red dye Sugar fermentation& H2S production

XLDagar

Xylose, lysine, Fe,

thiosulfate, phenolred

Enterobacter,Escherichia,

Proteus,Providencia,

Salmonella, Shigella

Functional Type:MISCELLANEOUS



1. ANAEROBIC GROWTHThioglycollate broth

Mannitol Salt Agar

Lactose broth

2. SPECIMEN TRANSPORT

Stuart’s medium

Amie’s medium

Functional Type:MISCELLANEOUS



C. ASSAY• Mueller – Hinton agar

• BAP

D. ENUMERATION

• Nutrient Agar

• Chromocult Coliform Agar • Differential Coliform Agar

INOCULATIONOF CULTURE MEDIA



A. TUBED MEDIA LIQUID

SEMI – SOLID

SOLID

B. PLATED MEDIA



Preserving Bacterial Cultures



Figure 6.10a, b

Deep-freezing

Lyophilization:

Frozen then vacuum-dehydrated

III. BIOCHEMICAL TEST



Forms the precise basis for bacterial

species identification

Designed to demonstrate the enzymatic

system within a bacterial cell

Fermentation Pathways



A. CARBOHYDRATE

FERMENTATION TEST



CULTURE MEDIA COMPOSITION

Triple Sugar Iron

Kligler’s Iron Agar

Russel’s Double Agar

TSI: Determines if a GNB utilizes glucose, lactose or sucrose fermentatively & forms H2S.

Triple Sugar Iron Agar

• INDICATORS :



C O S

A. Phenol Red

• phenolsulfonphthalein (PSP)

B. Ferrous Ammonium Sulfate

• H2S indicator

• (+) blackening of the medium

TSI REACTIONS: (Slant over Butt)K = alkaline; NC = No change; H2S = blackening

A = acidic; Gas = bubbles, in agar



cracks in agar SLANT/BUTTREACTION

COLOR SUGARFERMENTED

K/K; K/NC RED/RED

K/A RED/YELLOW

A/A YELLOW/

YELLOWA/K YELLOW/RED

TSI : CarbohydrateFermentation Test



• Quality Control

A/A Gas+: Escherichia coli

K/A H2S+: Salmonella typhi

K/NC or K/K: Pseudomonas aeruginosa

B. IMViC TEST

TESTS:



INDOLE

METHYL RED

VOGUES – PROSKAUER TEST

CITRATE

MEDIA USED:

• Tryptophan Broth or SIM• MRVP or Clark & Lubs Dextrose Broth

• Simmon’s Citrate Slant

tryptophanase



Tryptophan pyruvic acid, NH3 indole

METHODS:• Kovac’s: add p –dimethylaminobenzaldehyde

• Ehrlich’s: add ether/xylene + pdab

• Spot Indole Test: 1% p-dimethylaminocinnamaldehyde

INDOLE TESTQUALITY CONTROL:



A.KOVAC’S method (+): E. coli (-): K. pneumoniae

B. EHRLICH’S method (+): Elizabethkingia

meningoseptica

(-): Paracoccus yeeii sp.nov(CDC group EO-2)

2. METHYL RED / VOGES-PROSKAUER



Determines an organism’s ability to… produce & maintain stable acid end products

produce neutral end products

QUALITY CONTROL:

METHYL RED

(+): E. coli (-): Enterobacter

cloacae

VOGES-PROSKAUER

(+): E. cloacae (-): E. coli

METHYL RED TEST

GLUCOSE large amount of acidFermentation



METHODS:

•MRVP medium•Buffered Peptone Glucose Broth

VOGES – PROSKAUER TEST

GLUCOSE Acids & add



fermentation acetoin α-naphtholand KOH

Barritt’s method for GNB:

•Solution A ( α -naphthol)

•Solution B (KOH)

Sodiumutilize produce

3. CITRATE TEST



MEDIUM: Simmon’s Citrate slant

pH INDICATOR: Bromthymol Blue

(+) prussian blue (-) yellow

green

citrate &inorganicNH4 salts

C. CATALASE

• Differentiates Staphylococcus species from



Streptococcus species

30% H202 O2 + H2O

METHOD:

• Place a drop of 30% H2O2 onto the slide

• Mix in the inoculum.

catalase

D. OXIDASE TEST



• To determine the presence of ____________________by oxidation

of oxidase reagent to __________ , a

____________-colored end product.

• Detects the ability of organisms to

oxidize aromatic amines in thepresence of air.

Oxidase reagent.

(aromatic amines)

Cytochrome

oxidase

IODOPHENOL



REAGENTS:

PADAM

para-aminodimethyl aniline monohydrochloride

TMPDD

1% tetra-methyl para-phenylenediaminedihydrochloride

Spot Oxidase Test = 10 sec

UREA

urease

NH3 CO2 H2O

E. UREA HYDROLYSIS



MEDIUM: Christensen’s Urea Agar

pH INDICATOR: phenol red

From light orange (pH 6.8) to magenta (pH 8.1)

UREA NH3 + CO2 + H2O

Ph l l i

F. PHENYLALANINE DEAMINASETEST

Ph l i



• REAGENT: 10 % Ferric chloride

Phenylalanine deaminase

Phenylalanine Phenylpyruvicacid

Add 4-5 drops of

10 % FeCl3

G. LYSINE IRON AGAR TEST

LysineLysine

decarboxylaseCadaverine Lysine



• MEDIA: Lysine Iron Agar

• pH INDICATOR: Bromcresol purple • H2S indicator: Ferrous NH 4 citrate • Sodium thiosulfate

LysineLysine

deaminaseFerrous NH4 citrate,

Flavin mononucleotide

Compd. (NH3)

SEROLOGICAL TESTING

I l ti d tib d ti i



• Involves antigen and antibody reaction invitro using the patients serum

Examples:ASO

RPR

Widal Test

ANIMAL INOCULATION

k f t f i l li



• makes use of some types of animal as livemedium for cultivation

• EXAMPLES: – armadillo and mouse’s foot pads for the

isolation of Mycobacterium leprae

Documents

Bacte Prelim Lecnotes 2012