MT 302 Lec- Bacteriology

History of Microbiology
Discovery of Microorganism
Lucretius (98-55 B.C) and Girolamo (1478-1553)

 “Disease is caused by Invisible living creature”

 “de Contagione” in 1546 written by Girolamo Fracastoro which he states that free living
organism cause the disease
 In his poem entitled “Syphilis sive Morbus Gallicus” (translated “Syphilis or the French
Disease”) he described in remarkably accurate, yet mythical, poetic detail the clinical
consequences of syphilis.
Antonie van Leeuwenhoek (1632–1723)

 Father of Microbiology
 First identifying microorganisms, or “little animals,” or animacules using his newly developed
microscope in 1677, thereby confirming Fracastoro’s hypothesis
 “Father of Bacteriology and Protozoology”
 Animacules is later given a name Microbes by Sedillot (1878)
Spontaneous Generation
Aristotle (384-322 B.C.)

 “Living organisms could develop from non-living materials”

Contradict the Spontaneous Theory
John Needham (1731-1781)

 He proposed that tiny organism(animalcules) arose spontaneously on the mutton gravy.

 He covered the flasks with cork as done by Redi, Still the microbes appeared on mutton broth.
Francesco Redi

 Demonstrate that maggot could not arise spontaneously from a decaying meat.
Lazzaro Spallanzai (1729 – 1799)

 He demonstrated that air carried germs to the culture medium.

 He showed that boiled broth would not give rise to microscopic forms of life
Biogenesis

 Living cell can arise from a preexisting living cell.

Rudolf Virchow

 Challenge the spontaneous generation with the concept of biogenesis

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Theodor Schwann

 No growth on flask with nutrient agar when pass thru a high temp
Heinrich Schroeder and Theodore von Dusch

 When sterile cotton is placed on the flask of heat sterilized medium

John Tyndall

 Introduce the use of moist heat for 3 consecutive days to eradicate endospores and vegetative
cells
Louis Pasteur

 Father of Medical Microbiology

 Father of Modern Microbiology
 He proposed the used of heat can kill microorganism that is known as ANTISEPTIC
 Disapprove the spontaneous generation,
 Developed vaccine against rabies and anthrax
 Improve the wine making by fermentation and pasteurization
Antisepsis
Ignaz Semmelweis

 Demonstrate hand washing

 He demonstrated the routine handwashing can prevent the spread of disease.
Joseph Lister

 Introduce antiseptic surgery

 Introduce handwashing and the use of phenol as an antimicrobial agent for surgical wound
dressing to British surgery
Germ Theory
Robert Koch

 Show proof that bacteria cause disease

 Discover Bacillus anthracis
 Discover Mycobacterium tuberculosis
 First to cultivate bacteria on Boiled potato, gelatin and meat extract
Vaccination
Edward Jenner

 Introduced the concept of vaccination

Louis Pasteur
Emil von Behring
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 He prepared antitoxins for diphtheria and tetanus

Elie Metchnikoff

 Describe phagocytosis

Introduction to Microbiology-
Study of Microorganism – Bacteria, Fungi, Parasites, Viruses, Prions
Organism can be identified as:

 Prokaryotes - “pro” before and “Karyon” means nucleus, nuts or kernel

o Consist of single close compartment
o Lacks define nucleus
o Simple internal organization.
o Examples: Bacteria, cyanobacteria, Blue Algae, Archaeabacteria
 Cyanobacteria AKA Blue-green bacteria
 Has elaborate cytoplasmic membrane which is the thylakoid for
photosynthesis
 Capable of Nitrogen fixation
 Bacteria cells
 Prokaryotes, lacking welldefined nuclei and membrane-bound
organelles, and with chromosomes composed of a single closed DNA
circle
 Eukaryotes - “EU” means True
o a defined membrane-bound nucleus
o Extensive internal membranes that enclose other compartment, Organelles
o Examples: Animals, Fungi, Protozoans, Parasite
 Fungi
 Cell Membrane contains Sterols
 Cell wall contains Glucans
 Cell can be Yeast or Molds
 Example: Candida albicans, Aspergillus spp.
 Plant Cell
 Contain additional organelles like
 Chloroplast: sites of photosynthesis
Archaebacteria (“archaics”-ancient) aka extremist

 Extremophiles
 Found in the drainage fluids of abandoned mine shafts); and thermophiles (prokaryotes that live
at very high temperatures)
 Do not contain peptidoglycans but contain protein/glycoprotein walls known as “S layer”
 Examples: Methanospirillium, Halobacterium and Sulfolobus
Viruses

 Viruses are either DNA or RNA

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 Viruses are acellular

 Viruses are obligate intracellular parasites
 Examples: HIV, SARS Cov 2, Tobacco Mosaic virus, Bactreiophage
Bacterial Taxonomy

 Classification- organization of organism with similar morphologic, physiologic and genetic traits
 Carl von Linne - Swedish botanist that laid down rules for taxonomy.
Bacterial Cell

 Cell Envelope
o The outermost structure of bacterial cell
 Plasma Membrane
 A phospholipid bilayer with embedded proteins that envelope the
cytoplasm
 Act as osmotic barrier
 Electron transport chain where energy is generated
 Cell Wall
 Outermost layer of the cell
 Consist of peptidoglycans or murein layer
 Primary target of antimicrobial agent
 Prevent bacterial cell rupture
 Serve as point of anchorage for flagella
 Helps in staining characteristics
 Gram Positive Cell Wall
 Peptidoglycan or murein layer consists of glycan (polysaccharide) chains
of alternating N-acetylDglucosamine (NAG) and N-acetyl Dmuramic
acid (NAM)
 teichoic acid (anchored to the peptidoglycan)
 lipoteichoic acid (anchored to the plasma membrane).
 Examples: Staphylococcus spp, Streptococcus spp
 Gram Negative Cell Wall
 Outer membrane
o Proteins, Phospholipids and Lipopolysaccharide (LPS)
 Lipid A (produce Endotoxins)
 Core Polysaccharide
 Antigenic O
o Negatively charge and evades phagocytosis
o Allows Hydrophilic compounds enter the cell thru porins
o Barrier of toxic substances
o Examples: Enterobacteriaceae spp
 Acid Fast Cell Wall
 Peptidoglycan
 Mycolic acid (glycolipids and fatty acids
o Strong hydrophobic
 Examples: Nocardia, Mycobacterium spp
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 No Cell Wall
 Contains Sterols like in Fungi
 Examples: Mycoplasma, Ureaplasma

Cytoplasmic Structure

 Ribosomes
o Site of Protein synthesis
o Consist of RNA and Protein
o 70S in size with 2 subunits: 50s and 30s
 Genome
o A single, circular chromosome
o A diffused nucleoid or chromatin body that is attached to a mesosome
 Plasmid
o Extrachromosomal double stranded DNA
o Located at the cytoplasm
o Site for gene encode and antibiotic resistance and toxin production
o Large plasmid (prod beta lactamase) or small plasmid (resistant to tetracycline and
chloramphenicol)
 Inclusion bodies
o serve as a energy source or food reserve of the bacteria
o Mainly Polysaccharides which lessen the osmotic pressure
o Examples Polyphosphate granules of Corynebacteria diphtheriae (Babes-Ernst bodies)
Bipolar bodies of Yersinia pestis Much granules of Mycobacterium tuberculosis
 Endospores/ Asexual spores
o Small dormant structure outside the bacterial cell
o Aid for the survival of the bacteria
o Composed of calcium dipicolinate (dipicolinic acid and calcium ions)
o Examples: Bacillus and Clostridium spp
o Terminal spores: Clostridium tetani
o Subterminal spores: Clostridium botulinum
o Central spores: Bacillus anthracis
 Glycocalyx
o Outward complex of polysaccharide on the bacteria.
o For the attachment to the surface of object or tissue
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o Appear as capsule or slime layer

 Capsule
o Polysaccharide polymer
o Act as a virulence factor that inhibit the phagocytosis.
o Can be used in the identification
o Example: Streptococcus pneumoniae, Klebsiella pneumoniae and Salmonella typhi
 Slime Layer
o Unorganized material that is loosely attached to the cell
o Made up of polysaccharides
o Aid for the adherence to objects and also resist phagocytosis
 Flagella
o Exterior protein filaments (flagellin) that rotate and cause the motility of the bacteria.
o Arrangement of flagella:
 Atrichous – No flagella, non-motile
 Monotrichous/polar flagella – Single flagella at one pole, e.g Vibrio spp.
 Amphitrichous – single flagellum at both ends
 Lophotrichous- tuft or group of flagella on one end or both end
 Peritrichous- flagella all over the cell, E.g Enterobacteriaceae except Klebsiella
spp & Shigella spp.
 Sex pili
o Conjugation pili
o Non motile, hollow protein that connects two bacterial cell and mediate DNA exchange
 Fimbriae or Somatic pilus
o Non flagellar, sticky, hairlike appendages that adhere to one another or to the
environment
o Example: Neisseria gonorrheae, Pseudomonas spp

Microbial Growth
Three major needs for growth

 Carbon (50%)
 Nitrogen (14%)
 Source of Energy (Adenosine Triphosphate, ATP)
 Smaller amounts of molecules
o phosphate for nucleic acids
o phospholipids of cell membranes
o Sulfur for protein synthesis

Nutritional requirements for growth

 Autotrophs (Lithotrophs)
o Using carbon dioxide as a sole source of carbon addition with water and inorganic salts
o Obtain energy by
 photosynthetically (phototrophs)
 Oxidation of inorganic compounds (chemolithotrophs)
 Heterotrophs
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o Requires more complex substances.

o Require organic source of carbon (e.g Glucose) and energy by oxidizing or fermenting
organic substances.
o All bacteria in the human body are heterotroph.
o Examples: Escherichia coli and Pseudomonas aeruginosa

Environmental Factors Affecting Growth

 pH
oPathogenic organism best grown in NEUTRAL pH
opH of the media used inside the lab: 7.0 -7.5
oNeutrophile
 Between pH 6.5 – 7.5
 Clinically significant
o Acidophiles
 Best in acidic habitats
 Example: Helicobacter pylori, Sulfolubus,
o Akalinophiles
 Live in alkaline soils and water up to pH 11.5
 Example: Vibrio cholerae
 Temperature
o Influence the rate if growth of a bacterial culture
o Psychrophiles
 Bacteria grow best in cold temperature
 10 to 20 Celsius
o Mesophiles
 Bacterial that grow in optimal temperature
 20 to 40 Celsius
o Thermophiles
 Bacterial at high temperatures
 50 to 60 Celsius
Atmospheric requirements for Growth

 Obligate Aerobes
o Requires oxygen for growth
o Example: Brucella spp., Bordetella spp., Mycobacteria spp., Pseudomonas spp
 Aerotolerant anaerobes (Facultative aerobes)
o Can survive in the presence of oxygen but do not used oxygen in metabolism.
o Examples: Clostridium spp., Proprionobacterium spp.
 Obligate Anaerobes
o Cannot grow in the presence of Oxygen
o Example: Bacteroides fragilis
 Facultative anaerobes
o Can grow either with or without oxygen
o Examples: Escherichia coli
 Capnophilic
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o Grow best when the atmosphere is enriched with extra Carbon dioxide (5% to 10%)
o Examples: Hemophilus influenzae, Streptococcus pneumoniae
 Microaerophilic
o Bacteria require a reduced level of oxygen to grow
o Examples: Campylobacter spp. (5% to 6%)

Bacterial Growth

 Bacterial replicates in Binary fission

 Generation time (doubling time)
 Time required for one cell to divide into two.
 Fast growing: Escherichia coli (20 minutes)
 Slow growing: Mycobacterium tuberculosis (24 hours)
o Lag phase
 No cell division
 Bacteria is preparing to divide
o Log phase (Exponential Phase)
 Bacteria numbers increase logarithmically
o Stationary phase
 Nutrient in this phase becoming limited and the number bacteria remain constant
o Death phase
 Number of nonviable bacterial cells exceed number of viable cells
Determination of Cell Number

 Direct Counting under Microscope

o used to estimate the number of bacteria present in a specimen.
 Direct Plate Counting
o This method provides a count of viable cells only.
o Colony Forming unit per milliliter (CFU/mL)
o For Bacterial cell count in urine sample and water sample.
 Density measurement
o Refer as cloudiness or turbidity
o Density of a bacterial broth culture in log phase can be correlated to CFU/mL of the
culture.
o Used to prepare a standard inoculum for antimicrobial susceptibility testing

Microbial Metabolism
Bacterial Metabolism
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 Consist of Biochemical Reactions use to breakdown organic compounds and the reactions they
use to synthesize new bacterial parts.
 Use for Diagnostic scheme
o By utilizing various substrate as a carbon source.
o Production of various substrates ○ Production of acid or alkaline pH
 Catabolism – breakdown of Carbohydrates and produce energy
 Glucose is the essential nutrient for energy production
Two general processes of producing energy from glucose

 Respiration (Oxidation)
o An efficient energy generating process in which molecular oxygen is the final electron
acceptor. Glucose → Carbon dioxide and Water
o Obligate aerobes and facultative anaerobes: Aerobic respiration (Oxygen)
o Anaerobic: Anaerobic respiration in the forms of inorganic forms (nitrate and sulfate)

o Three Major Metabolic Pathways (Glucose to Pyruvic Acid)

 Embeden Meyerhof Parnas Glycolytic Pathway (Glcolysis)
 Major pathway in conversion of glucose to pyruvate
 Major route of glucose metabolism
 Generates reducing power in the form of NADH2
 Generates energy in the form of ATP
 Anaerobic; does not require oxygen
 Used by many bacteria, including all members of Enterobacteriaceae
 Pentose Phosphate Pathway
 Alternative to EMP pathway for carbohydrate metabolism
 Conversion of glucose to ribulose-5-phosphate, which is rearranged into
other 3-, 4-, 5-, 6-, and 7-carbon sugars
 Provides pentoses for nucleotide synthesis
 Produces glyceraldehyde-3-phosphate, which can be converted to
pyruvate
 Used by heterolactic fermenting bacteria, such as Lactobacilli spp. and
Brucella abortus
 Entner-Doudoroff Pathway
 Converts glucose-6-phosphate to pyruvate and glyceraldehyde phosphate
 Generates one NADPH per molecule of glucose but uses one ATP
 Used by Pseudomonas, Alcaligenes, Enterococcus faecalis, and other
bacteria lacking.
o Aerobic Utilization of Pyruvate (Oxidation)
 Krebs cycle or Tricarboxylic acid (TCA) cycle
 A series of eight enzymatically catalyzed reactions that transfer much of
this stored energy to the coenzymes NAD+ and FAD.
 This cycle results in the production of citric acid and the evolution of
carbon dioxide
 Fermentation
o Anaerobic process by obligate and facultative anaerobes
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o Electron acceptor: Organic compound

o Substrate is not completely reduced or released.
o End product will accumulate at the medium.
o Example of test: Voges Proskauer and Methyl Red
o Sugar + yeast- oxygen= CO2 and Alcohol
o Fermentattion Pathway: Anaerobic Utilization of Pyruvic Acid
 Alcoholic fermentation
 Glucose to ethanol
 Homolactic fermentation
o Glucose to Lactic acid
o E.g., Streptococcus and Lactobacillus ○ Yogurt, Pickles, sauerkraut
 Heterolactic
o End product: Lactic acid and others
o Carbon dioxide, alcohols, formic acid, acetic acid

 Proprionic Fermentation
o Proprionic acid
o Proprionibacterium acnes
 Mixed Acid fermentation
o Sugar fermentation and acids like lactic acid, acetic acid, succinic acid
and formic acid
o Enterobacteriaceae, Escherichia coli, Shigella and Salmonella spp.
 Butanediol Fermentation
o End product: Acetoin and 2,3 butanediol
o Klebsiella spp, Enterobacter and Serratia spp., Voges Proskauer
 Butyric Fermentation
o Butyric acid as end product
o Obligate Anaerobes: Clostridium, Fusobacterium and Eubacterium spp.

Carbohydrate Utilization and Lactose Fermentation

 Fermentation of the sugar is usually detected by acid production and a concomitant change of
color resulting from a pH indicator present in the culture medium.
 Lactose
o a disaccharide consisting of one molecule of glucose and one molecule of galactose
linked together by a galactoside bond.
Bacterial Genetics
History

 Frederick Miescher (1869)

o First discover DNA
 Phoebus A. T Levine
o Discovered DNA contains phosphate and 5 carbon sugar (pentose) and nitrogen bases
 Rosalind Franklin
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o Discover that DNA is a helical structure by X-Ray crystallography

 James Watson and Francis crick (1950)
o 3D structure of DNA

Structure and function of Genetic Material

 DNA=deoxyribonucleic acid
 RNA= ibonucleic acid
 Basic building blocks:
o Nucleotides: Phosphate group, pentose, sugar, nitrogen

Structure of DNA

 Double stranded (double helix)

 Chains of nucleotides
 5’ TO 3’ (strands are anti-parallel)
 Complimentary base pairing
o Adenine (Purine)-Thymine (Pyrimidine)
o Guanine (Purine)-Cytosine (Pyrimidine)

Bacterial Genome

 Bacteria have closed, circular DNA

 Genome or chromosomes
o Genetic material in an organism
o It contains all the information needed for cell growth and replication.
 Genes
o Specific DNA sequences that code for the amino acid sequence in one protein
 E. Coli
o 4 million base pairs
o 1 mm long (over 1000 times larger than actual bacterial cell)
o DNA takes up around 10% of cell volume

Extrachromosomal Elements

 Plasmids
o Extrachromosomal double stranded DNA
o Not essential for bacterial growth
o Code for antibiotic resistance
o Located in the cytoplasm of the cell and are self-replicating and passed to daughter cells,
similar to chromosomal DNA
o Can transfer gene through conjugation

Jumping Genes

 Pieces of DNA is mobile and may jump from one place in the chromosome to another place
 Insertion sequence
o Odes for only one gene, a transposase enzyme that allows the is element to pop into and
out of DNA
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 Transposons
o Mobile elements that contain additional genes.
o Carry antibiotic resistance genes

DNA Replication Occurs at the Replication Fork

 • 5’ TO 3 ‘
 DNA helicase-unzips + parental DNA strand that is used as a template
o Leading stand (5’ to 3’-continuous)
 DNA polymerase-joins growing DNA strand after nucleotides are aligned
(complimentary)
o Lagging strand (5’ to 3’-not continuous)
 RNA polymerase (makes short RNA primer)
 DNA polymerase (extends RNA primer then digests RNA primer and replaces it
with DNA)
 DNA ligase (seals okazaki fragments-the newly formed DNA fragments)
Protein Synthesis
DNA-(transcription)- mRNA-(translation)-protein

Transcription

 One strand of DNA used as a template to make a complimentary strand of mRNA

 Promoter/RNA polymerase/termination site/5’ to 3’
 Ways in which RNA & DNA differ:
o RNA is SS
o RNA sugar is ribose
o Base pairing-A-U

Types of RNA

 mRNA: messenger RNA

o contains 3 bases (codon)
 rRNA: ribosomal RNA
o Comprises the 70 S ribosome
 TRNA: transfer RNA
o Transfers amino acids to ribosomes for protein synthesis
o Contains the anticodon (3 base sequence that is complimentary to codon on mRNA)

Genetic Code

 DNA: triplet code

 mRNA: codon (complimentary to triplet code of dna)
 tRNA: anticodon (complimentary to codon)
 Codons: code for the production of a specific amino acid
 20 amino acids
 3 base code
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 Degenerative: more than 1 codon codes for an amino acid

 Universal: in all living organisms
Translation

 Three parts:
o Initiation-start codon (AUG)
o Elongation-ribosome moves along mRNA
o Termination: stop codon reached/polypeptide released and new protein forms
 rRNA=subunits that form the 70 s ribosomes (protein synthesis occurs here)
 tRNA=transfers amino acids to ribosomes for protein synthesis)
Mechanism of change of bacterial genetic information

 Mutation
o Changes in base sequence of DNA/lethal and inheritable
o Can be:
 Harmful
 Lethal
 Helpful
 Silent

 Recombination
o A method by which genes are transferred or exchanged between homologous (similar)
regions on two DNA molecules
Restriction Enzymes

 Restrict the replication of viral genome

 Cut the DNA sequence
 Methylation can inhibit the cutting of restriction enzymes
 First three letters in the restriction endonuclease name indicate the bacterial source of the enzyme
 Examples:
o Ecori was isolated from E. Coli,
o enzyme Hindiii from H. Influenzae

Genetic Transfer in Bacteria

 Genetic transfer-results in genetic variation

 Genetic variation-needed for evolution
 Three ways:
o Transformation: genes transferred from one bacterium to another as “naked” DNA
o Conjugation: plasmids transferred 1 bacterium to another via a pilus
o Transduction: DNA transferred from 1 bacterium to another by a virus

Transformation

 The uptake and incorporation of naked DNA into a bacterial cell

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 Then recombination is done

 Streptococcus pneumoniae, Neisseria gonorrhoeae, and H. influenzae
 Escherichia coli is used only in production of insulin
Transduction

 The transfer of bacterial genes by a bacteriophage (virus-infected bacterium) from one cell to
another
 Bacteriophage
o A chromosome (DNA or RNA) surrounded by a protein coat.
o Can undergo lytic pathway
 Lysogeny
o Phage DNA instead becomes incorporated into the bacterial genome and replicate with
the bacterial chromosome
o Phage known as this stage temperate
 Example: Corynebacterium diphtheriae carrying the diphtheria toxin
Conjugation

 The transfer of genetic material from a donor bacterial strain to a recipient strain.
 By sex pilus the f+ binds to the f-
 Plasmid and chromosomal gene are transferred.
 High frequency strains
o When the f factor is integrated into the bacterial chromosome rather than a plasmid, there
is a higher frequency of transfer of adjacent bacterial chromosomal genes.
Microscope
Microscopy

 The use of light or electrons to magnify object

 Invented by Leeuwenhoek
 Greek “micro” means “small” and “skopein” meaning “to view”
General Principles of Microscopy

 Wave length radiation

o The distance between two corresponding parts of wave
 Magnification
o The apparent increase in size of an object results when a beam of radiation refracts
(bends) as it passes through a lens.
o Curved glass refracts lights
o Magnetic field refracts electron beam.
 Empty magnification
o When an image is magnified in a million times but a faint ang blurry.
 Determine the clarity of magnification
o Resolution (resolving power)- the ability to distinguish objects that are close together.
 Resolution distance is dependent on:
 Wavelength of electromagnetic radiation
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 Numerical aperture of lens- ability of the lens to gather light.

o Contrast- staining the organism
 Differences in intensity between two objects or between an object and its
background.
 Microorganism has a little contrast.
 Staining technique- to increase the contrast (Acid fast stain, Gram stain)
Light Microscope

 Common microscope used in laboratory.

 Bright Field Microscope
o Simple microscope
 Contains a single magnifying lens
 Like magnifying glass
 More n the light and contrast
 Compound Microscope
o Uses series of lenses for magnification.
 Objectives
 Ocular lenses
o Galileo Galilei already made compound microscope early 1590

Parts of the Microscope

 Ocular lens/ Eyepiece lens

o Remagnifies the image formed by the objective lens.
o 10x or 15x power.
 Tube
o Connects the eyepiece to the objective lens.
 Body
o Transmits the image from the objective lens to the ocular lens using prisms.
 Arm
o Supports the tube and connects it to the base.
 Objective lens
o Primary lenses that magnify the specimen. (3 or 4 OL)
o The shorter the lens the lower the power.
o High power objective lens is retractable (40XR). If hit by the slide it pushes to protect
lens and slide.
 Scanning Objective lens- 4x (red)
 Low power objective lens- 10x (yellow)
 High power objective lens- 40x (blue)
 Oil immersion objective lens- 100x (white)
 Stage
o Holds the microscope slide in position.
o Platform that places the slide.
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 Stage clip- hold the slide in place.

 Revolving nosepiece or turret
o Holds two or more objective lenses and can be rotated to easily change the power.
 Condenser
o Focus light through specimen. More useful to high power (400x and above)
 Diaphragm
o Controls the amount of light entering the condenser.
 Illuminator
o Light source
o Steady light source used in place of a mirror.
 Coarse focusing knob
o Moves the stage up and down to focus image.
 Fine focusing knob
 Base
o The bottom of the microscope, used for support.
 Rack stop
o Adjustment that determines how close the objective lens can get to the slide.

 Dark Field Microscope

o Utilize dark-filed stop in condenser that prevents light from directly entering the
objective lens.
o Only those light rays that are scattered by the specimen enter the objective lens.
o Useful for small or colorless cells
 Phase Microscope
o Trat one set of light rays differently from another set of light rays.
 In phase: with crest and through are aligned (brighter image).
 Out of phase: crest and through are not aligned (darker image)
o Phase plate
 Special filter which is mounted in a phase objective lens, retards these rays
another ¼ wavelength. So that they are ½ wavelength out of phase with their
neighbors.
 Phase Contrast Microscope
o Produce sharply defined image in which fine structures can be seen in living cells.
o Enhances the contrast between intracellular structures having slight differences in
refractive index.
o Ideal for observing flagella and cilia.
 Differential Interferences Contrast Microscope
o AKA Nomarski microscope
o Use prism that split light beams into their component wavelength (colors).
o Gives three dimensional or shadowed appearance.
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 Fluorescent Microscope
o Molecules that absorb energy from invisible radiation (such as ultraviolet light) and then
radiate the energy back as longer, visible wavelength.
o Uses an ultraviolet (UV) light source to fluorescent.
o Immunofluorescence
 Fluorescent dye are covalently linked to antibody that binds to the
complementary antigen.
 Example: Lyme disease, Rabies and Syphilis
o Pseudomonas aeruginosa are naturally fluorescent.
o Bacillus anthracis: Fluorescein isothiocyanate
o Mycobacterium tuberculosis: Auramine O
 Confocal Microscopy
o Use fluorescent dyes or fluorescent antibodies, but these microscopes uses ultraviolet
lasers to illuminate the fluorescent chemicals in only a single plane that is no thicker than
1.0 micrometer/
o Laser beam used to illuminate spots on specimen.
o Computer compiles images created from each point to generate a 3-dimensional image.
o Useful in biofilm assessment.
 Electron Microscope
o Provides detailed vies of the smallest bacteria, viruses, internal cellular structures, and
even molecules and large atoms.
o Ultrastructure
o 2 types:
 Transmission electron microscope
 Generates a beam of electron that ultimately produces an image on a
fluorescent screen.
 Dense area: Black electron (Dark area)
 Less dense: Screen fluoresces more brightly.
 Uses ultramicrotome (there is sectioning)
 Scanning electron microscope
 Uses magnetic fields within a vacuum tube to manipulate a beam of
electrons, primary called electrons.
 Electrons focuses them back and forth across the specimen’s surface,
which has previously been coated with a metal such as platinum or gold.
 Probe Microscopy
o Advance in microscopy utilizes minuscule, pointed, electronic probes to magnify more
than 100,000,000x.
o 2 variations:
 Scanning tunneling microscopes
 Passes a metallic probe back and forth across and slightly above the
surface of a specimen.
 Measures the flow of electrons to and from the probe and the specimen’s
surface.
 Tunneling current- amount of electron flow directly proportional to the
distance from the probe to the specimen’s surface.
 MT 302 Lec- Bacteriology

 Atomic force microscopes

 Uses a pointed probe.
 But it traverses the tip of the probe light on the surface of the specimen,
rather than at a distance.
 Atomic force microscope can magnify specimen that do not conduct
electrons.
Bacterial Morphology, Smear Preparation and Staining
Basic Shapes of Bacteria

 Cocci
o Staphylococci
 Irregular caster (grape-like structure)
o Micrococci
 Tertrads (4)
o Streptococci
 Chain pairs- (gram (+))

 Bacilli
o Coccobacilli
 Circular rod shape
o Bacilli
o Fusiform
 Linear
o Palisading
 Fence type
 Spirochetes
Fixation

 process by which internal and external structures are preserved and fixed in position.
 process by which organism is killed and firmly attached to microscope slide.
 Can’t differentiate organism because all violet
o Heat fixing
 preserves overall morphology but not internal structures.
o Chemical fixing (e. g Methanol)
 protects fine cellular substructure and morphology of larger, more delicate
organisms.
Simple Staining

 Use of one dye for staining.

 Dye is commonly basic dye
 Example is Crystal Violet
Differential Stains
Gram Staining
 MT 302 Lec- Bacteriology

 More than one dye is used to differentiate cells chemicals or structures.

 Developed by Hans Christian Gram (1884)
 Differentiates between two large groups of microorganisms
o Gram positive
o Gram negative
 Steps:
o Crystal violet
o Iodine
o Alcohol
o Safranin

Process Gram Positive Gram Negative

Crystal Violet Violet Violet
Iodine Violet Violet
Alcohol/Acetone Violet Red/Pink
(Decolorization)
Safranin Red Violet Red/Pink

Gram Positive Cell Wall

 Peptidoglycan is composed of alternating sugar molecules called N-acetylglucosamine (NAG)

and N-acetylmuramic acid (NAM).
 Has a thick layer of peptidoglycan
 Has Teichoic Acid
o Have negative electrical charges, which help give the surface of a Gram-positive
bacterium a negative charge and may play a role in the passage of ions through the wall.

 All cocci are gram positive except:

o Neisseria spp.
o Branhamella spp.
o Veilonella spp.

Gram Negative Cell

 Has a thin layer of peptidoglycan

 External wall membrane with a periplasmic space between.
o Lipopolysaccharide (LPS) which contains lipid A.
 All bacilli are gram negative except:
o Bacillus spp.
o Clostridium spp.
o Mycobacterium spp.
o Corynebacterium spp
o Listeria spp.
o Lactobacillus spp
o Proprionibacterium spp.
 MT 302 Lec- Bacteriology

o Erysipelothrix spp

Acid Fast Staining

 Franz Ziehl (1857–1926) and Friedrich Neelsen (1854–1894) in 1883

 used to stain bacteria that have a high lipid and wax content in their cell walls and do not stain
well with gram stain.
 Acid fast organism
o Nocardia spp.
o Rhodococcus
o Saccharomyces
o Cyclosporidium belli (Isospora belli)
o Mycobacterium spp

Process Ziehl Neelsen Kinyoun Modified Kinyoun

Primary Stain Carbol fuchsin Carbol fuchsin Carbol fuchsin

Mordant Heat Tergitol Tergitol

Decolorization 3% Acid Alcohol 3% Acid Alcohol 1% HSO4 and Alcohol

Secondary Stain Methylene Blue Methylene Blue Methylene Blue

Identify Nocardia and Cryptosporidia and

Mycobacterium Cyclospora
tuberculosis

Special Stain

 Fluorochrome
o Used to screen acid fast bacteria
o Example
 Acridine Orange
 fluorochrome dye that stains both gram positive and gram-negative
bacteria living or dead
 Bind to nucleic acid: ORANGE
 Calcofluor White
 a fluorochrome that binds to chitin in fungal cell walls.
 Metachromatic granules staining
o used to stain C. diphtheriae for observation of metachromatic granules
 Neisser
 MT 302 Lec- Bacteriology

o Methylene Blue and Crystal Violet

 Albert
o Malachite Green and Toluidine Blue
 Lactophenol blue
o used to stain the cell walls of medically important fungi grown in slide culture
 Negative (capsule) staining
o used primarily to reveal the presence of negatively charged bacterial capsules
o Mainly acid dyes
o Example: Eosin, Crystal Violet and nigrosin or India Ink
 Streptococcus pneumoniae
 Hemophilus influenzae
 Nesseria meningitides
 Bacillus anthracis
 Endospore stain
o For Clostridium and Bacillus spp.
o Endospores
 highly resistant cells form inside the cytoplasm of the bacteria and can survive
environmental extremes such as desiccation, heat, and harmful chemicals.

o Schaeffer-Fulton endospore stain

 uses heat to drive the primary stain, malachite green, into the endospore. After
cooling, the slide is decolorized with water and counterstained with safranin.
 Flagellar Stain
o Stain flagella
o Pararosaniline and carbolfuchsin, Leifson, Gray and Fisher (Primary stain)
o Mordants: tannic acid and potassium alum,
 Gray
 Carbol fuchsin and tannic acid
 Leifson
 Carbol fuchsin, Tannic Acid and Methylene Blue
o Periplasmic Flagella
 AKA Axial Filaments
 E.g Spirochetes
 Treponema spp.
 Borrelia spp.
 Leptospira spp.

Specimen Collection and Processing

Basic Specimen collection

 Specimen collect in acute phase and before antibiotic administration.

 Select correct anatomic site.
 Use proper technique in collecting and with minimal normal flora contamination.
 MT 302 Lec- Bacteriology

 Collect in appropriate quantity (not in qns.)

 Package the specimen in a container or transport medium designed to maintain the viability of
the organisms.
 Do not accept specimen with leakage.
 Proper labeling is a must.
 Notify the laboratory in advance if unusual pathogens or agents of bioterrorism are suspected
Lesion, Wounds or Abscesses

 Dacron/Polyester Swab
o Using Cotton swab can be toxic to the bacteria because of excessive fatty acids.
 Area should be cleansed before collection (avoid Normal Flora)
 Place in a sterile tube
 Advisable to have needle aspiration of the pus rather than the swab.
Nasopharyngeal and Pharyngeal Swab

 Direct to Selective Media (Blood Agar Plate)

 Hemophilus influenza, Neisseria meningitides and Bordetella pertussis

Sputum Collection

 Bronchoalveolar lavage
 Expectorate sputum: deep cough
 Induced sputum: aerosol induction
Collection of Sputum Specimen

 Instruct the patient as follows: Rinse mouth with tap water to remove food particles and debris.
 Have patient breathe deeply and cough several times to achieve a deep specimen.
 Patient should expectorate into dry, sterile container.
 Transport immediately at ambient temperature. Refrigerate if a delay of more than one hour is
anticipated.
 Collect 3 first morning sputum (For MTB)
 Quality of the specimen
o Volume
o Free from contaminant: Confirm using BARLETT’S CRITERIA
o Storage
o Transport
o Purulent, mucoid, bloody, saliva or induced sputum

Stool

 By rectal swab or collect stool sample

 Negative result not sufficient to exclude bacteria or parasite.
 Need of 3 specimens
 MT 302 Lec- Bacteriology

 Stool preservative: buffered formalin & polyvinyl alcohol w/ zinc

 Refrigerate 2 hours delay
Urine Specimen Collection

 Clean - Catch Midstream

o Least Invasive in collecting urine sample.
o First morning urine is advisable.
o Contamination can occur due to urethral contamination.
 Straight Catheterized Urine
o Slightly invasive
o Collection of the bladder urine.
o Risk of introducing urethral organism to the bladder (ascending route UTI)
o Procedure: Cleanse urethral area. Insert the catheter into bladder. Pass 15 mL urine then
collect remainder.
 Suprapubic Bladder Aspiration
o Introduction of the needle through the abdomen into bladder to collect urine sample.
o Bladder must be full before performing the procedure.
o For pediatric practice
o Procedure: Disinfect the skin. Needle aspiration above the symphysis pubis through the
abdominal wall to the full bladder.
 Indwelling Catheter or Folly Catheter
o Develop bacteruria that can cause predispose them to severe infection

Specimen Collection

 Specimen containers must be sterile (autoclave before using for the collection)
 Preferably urine that is remained in the bladder for 4 hours has a decreased chance of false
negative.
 Suprapubic bladder aspiration urine sample if to be tested for anaerobic organism must submitted
in syringe.
Specimen Transportation and Preservation

 Refrigeration of urine (4oC) for 24 hours if delay in processing of the specimen occur.
 Urine transport tube
o usually contains boric acid to preserve bacterial variability
o used only when refrigeration is not possible.
 Urine preservative tablet
o also contains boric acid as a preservative.
o dissolve in the urine if refrigeration is not possible after 2 hours.
 After collection, urine is transported at the laboratory as soon as possible.
 If the transport or processing of urine is delayed more than 2 hours, refrigerate the specimen.
 Repeat collection if the sample is greater than 2 hours old and if there is no proof of
refrigeration or preservation.
Blood Collection
 MT 302 Lec- Bacteriology

 Skin at the venipuncture site must be meticulously prepared using a bactericidal disinfectant: 2%
tincture of iodine, 10% polyvidone iodine, 70% alcohol, or 0.5% chlorhexidine in 70% alcohol.
 For Adult: 10 mL (5 mL blood: 50 mL Thioglycollate broth)
 At least two blood collection should be done.
 Sodium Polyanethol Sulfonate (SPS)
o an anticoagulant is recommended because it also inhibits the antibacterial effect of serum
and phagocytes.
o Not greater than 0.025% SPS
o Also used for bone marrow or synovial fluid
o Other Anticoagulant: Heparin
o Not used: Citrate and Ethylenediamine Tetraacetic Acid
 Blood Culture Bottles
o Mostly with ARD (Antimicrobial Removal Device)
o Penicillinase: Penicillin
o Magnesium Sulfate: Tetracycline
o P-Aminobenzoic Acid: Sulfonamide

Transport Media

 Do not promote multiplication of microorganism but ensure preservation.

 STUART’S OR AMIE’S TRANSPORT
o upper respiratory tract specimens and fecal specimens
o For Fastidious organism
 Charcoal contained (AMIES)
o For Neisseria gonorrhea and Bordetella pertussis
 Cary and Blair
o Transport for Gram negative and anaerobe bacteria
 Jembec Transport or Transgrow
o James E Martin biological environmental chamber
o For transport of Neisseria gonorrhea and Moraxella spp.
o Contained Sodium Bicarbonate and Sodium Citrate

Specimen Storage

 Room Temperature Only

o Cerebrospinal fluid: 6 hours at incubator
o Specimen for anaerobic determination
o Stool sample for Clostridium difficile
 Refrigeration at 4 oC
o Urine
o Stool
o Sputum
o Swab
o Catheters
o Viral specimen
 Level of Priority
 MT 302 Lec- Bacteriology

o Level 1
 classified as critical because they represent a potentially life-threatening illness
and are from an invasive source.
 Require immediate processing
o Level 2
 unprotected and may quickly degrade or have overgrowth of contaminating flora.
 Mainly fastidious
o Level 3
 Requires quantitation
o Level 4
 Specimens that arrive in transport or holding media

 Reject Specimen
o The information on the label does not match the information on the requisition or the
specimen is not labeled at all.
o The specimen has been transported at the improper temperature.
o The specimen has not been transported in the proper medium (e.g., specimens for
anaerobic bacteria submitted in aerobic transports).
o The quantity of specimen is insufficient for testing (the specimen is considered quantity
not sufficient [QNS]).
o The specimen is leaking.
o The specimen transport time exceeds 2 hours post collection or the specimen is not
preserved.
o The specimen was received in a fixative (formalin), which, in essence, kills any
microorganism present.
o The specimen has been received for anaerobic culture from a site known to have
anaerobes as part of the normal flora (vagina, mouth).
o The specimen is dried.
o Processing the specimen would produce information of questionable medical value (e.g.,
Foley catheter tip)
o More than one specimen is submitted for one day. Except for sputum
o One swab submitted in multi-le request for various organism.
 MT 302 Lec- Bacteriology

 Observation
o Macroscopic
 Swab or aspirate
 Stool consistency (formed or liquid, bloody or mucus)
 Volume
 Fluid: clear or cloudy
o Microscopic
 Sputum
 Give technologist and physician of infectious process
 Guide for work up
 Dictate the need for non-routine or additional testing
Disinfection and Sterilization
Srerilization

 Refers to the destruction of all forms of life, including bacterial spores.

 Chemical or physical method.
Disinfection

 Chemical agents applied to inanimate objects.

Antiseptic

 A substance applied to the skin for the purpose of eliminating or reducting the number of bacteria
present.
 Do not kill spores

Factors that influence the degree of killing

 Types of organism
o Organism like lipid viruses, bacteria are least resistance to disinfectant like alcohol.
o More Resistant to Least Resistant
 Prions
 Naked pieces of protein, similar to a virus but without the nucleic acid.
 121 oC for several hours while immersed in acid or basic solutions.
 Bacterial spores
 Mycobacteria
 Nonlipid viruses
 Fungi
 Bacteria
 Lipid viruses
 Different to lipid containing bacteria which is Mycobacterium spp.
 Concentration of disinfecting agent
o Proper concentration of disinfecting agents ensures the inactivation of target organisms
and promote safe and cost-effective practices.
o Example:
 MT 302 Lec- Bacteriology

 Povidone iodine: concentrated

 Sodium hypochlorite: 1:10 dilution
 Nature of surface to be disinfected
o Depends o the surface if it is smooth, it is easy to disinfect and rough surface will be hard
to disinfect.
 Temperature
o Generally used at room temperature (20 oC to 22 oC)
o Work faster in room temp than in Cold temperature.
 Number of organisms
o Microbial load
 Total number of organisms
 Exposure time that is necessary for 99.9% elimination of microorganisms.
o Higher numbers of organisms require longer exposure times.
 Prescence of organic material
o Blood, mucus, and pus can inactivate the disinfecting agent.
o Bleach is easily inactivated by organic materials.
o Instruments and surfaces should be cleansed of excess organic material before
disinfection.
 Contact time
o The amount of time of disinfectant or sterilant to be in the surface.
o A function not only of the agent itself but also of the bioburden on the object, the type of
microorganism that is to be killed, and the presence of organic material and temperature
at which the agent is being used.
o A table in the laboratory need to have a contact time of 10-15 minutes.
 pH
o it is critical to make sure at what pH the agent is active and what the pH of the material to
be exposed to the agent is at the time the process will be done.
o Use freshly tetride bleach solution when pH drops it will inactivate bleach

Biofilm

 A community of bacteria or other microorganism

 Can be seen in Catheters, pipes
 Not easy to disinfect
Quorom testing

 Communication mechanism between bacteria that allows specific processes to be controlled.

 Bacteria produce and release chemical signal molecules called autoinducers that increase in
concentration as a function of cell density.
Three Devices Classification

 Critical materials
o Materials that invade sterile tissue or enter vascular system.
o Requires sterilization
 Semi Critical Materials
o Materials come into contact with mucous membrane
 MT 302 Lec- Bacteriology

o Require high- level disinfection agent

 Non critical Materials
o Require intermediate- level to low- level disinfection before contact with skin.

Physical Method

 Heat
o Most common used for the elimination of microorganism.
o Moist heat
 Autoclave
 Principle: heat under pressure
 Aal microorganisms (except prions) and their endospores are destroyed
within approximately 15 minutes of exposure
 121o C per 15 psi
o Dry heat
 This is the method used for heat stable substances that are not penetrated by
moist heat, such as oils.
 For sterilizing of glass wares
 Examples
 Ovens
o Operated between 50o C to 250/300o C
o Holding period of 160 0C to 170 for 1 to 2 hours is desirable.
 Boiling
o Methods that achieve disinfection but not sterilization; these
methods do not eliminate spores.
o At 100o C kills most organisms in approximately 10 minutes.
o Kills vegetative forms
 Inoculating loop and needle sterilizer

Pasteurization

 Eliminates food-borne pathogens and organisms responsible for food spoilage

 It is performed at 63o C for 30 minutes.
 Holder method or low temperature holding/ batch method
o Milk is heated 63 oC for 3 minutes
 Flash process/ high temperature/ short time
o At 72o C for 15-20 sec. Rapid cooling to 13o C
 Ultrahigh temperature
o At 140 oC for 3 sec. cooled quickly in vacuum.

Filtration

 Used with both liquid and air

 Liquid
o Accomplished through the use of thin membrane filters composed of plastic polymers or
cellulose esters containing pores of certain size.
 MT 302 Lec- Bacteriology

o Bacteria, yeast, and molds: pores size of 0.45and 0.80um (allow pseudomonas like
organism)
o Membranes with pore size of 0.01um are capable of retaining small viruses.
o For antibiotic, vaccine, parental solutions.

Air

 Use of high efficiency particulate air (HEPA) filters.

 Able to remove microorganisms larger than 0.3micrometer.
 Used in laboratory and in room of immunocompromised patient
Radiation

 Ionizing
o In the form of gamma rays or electron beams, is short wavelength and high energy.
o Sed by the medical field for the sterilization of disposable supplies such as syringes,
catheters, and gloves.
 Nonionizing
o Form of ultraviolet rays is of long wavelength and low energy

Chemical method

 Chemosterilizer
o Chemicals agent may be used to sterilize
 Exert killing effect by:
o Reaction with components of the cytoplasmic membrane.
o Denaturation of cellular proteins.
o Reaction with thiol (-SH) groups of enzymes.
o Damaged of RNA and DNA.

Alcohol

 Ethyl alcohol and isopropyl alcohol

 Excellent in vitro bacterial (it kills microorganism) activity against most gram-positive and gram-
negative bacteria.
 Non sporicidal (can’t kill or endospores)
 Inactivated by the presence o organic material
 Antiseptic and disinfectant
Aldehyde

 Formaldehyde
o Aldehyde generally used as formalin, a 37% aqueous solution or formaldehyde gas.
o To disinfect biosafety hood.
o Potential carcinogenic.
o M.tuberculosis has been known to survive in many years to tissue fixed in formaldehyde.
 MT 302 Lec- Bacteriology

 Glutaraldehyde
o More, precisely, a saturated five-carbon dialdehyde that has broad spectrum activity and
rapid killing action and remains active n the presence of organic matter.
o Active in alkaline pH
o 2% germicidal in approximately 10 minutes and sporicidal in 3 to 10 hours.
o Principle: inactivation of DNA and RNA through alkylation of sulfhydryl and amino
groups.
o Bactericidal, pseudomonacidal, fungicidal, virucidal against HIV, and HBV with a
minimum of 10 minutes exposure at a temperature between 20 o C and 30 o C.
o Tuberculocidal
o Cold sterilant
 Iodine
o Tincture
 Alcohol and iodine solution, used mainly as antiseptic
o Iodophor
 Combination of iodine and neutral polymer carrier that increase the solubility of
the agent.
 Less irritating, nonstaining, and more stable than iodine in its pure form.
 Disinfectant and antiseptic
 Example is povidone iodine
 Contact time is greater than 30 seconds
o Principle: due to oxidative effects of molecular iodine (I2) hypoiodic acid (HOI)
 Chlorine
o Principle: based on the oxidative effects of hypochlorous acid, formed when chloride ions
are dissolved in water.
o Broad spectrum but used as sterilants because of the long exposure time required for
sporicidal action and their inactivation by organic matter.
o Influenced by the pH of the surrounding medium
o Stable for no longer than 30 days with 50% of the original concentration of chlorine
dissipating for 30 days.
o Contact time at least 3 minutes
o 1:10 dilution of 5.25% concentration of sodium hypochlorite is recommended.
 Quaternary ammonium compound
o Derived by substitution of four valence ammonium ion with alkayl halides.
o Principle:
 Reducing the surface tension of molecules in the liquid.
 Disruption of the cellular membrane, resulting in leakage of cell contents.
o Inactivated by organic matter and reduced by hard water and soap.
o Pseudomonas spp. grow at the quaternary ammonium compound
 Phenolics
o Ortho-phenylphenol and ortho-benzyl-para-chlorophenol
o Fairly broad spectrum of activity but are not sporicidal
o Principle: disruption of cell walls, resulting in precipitation of urine.
o Found in germicidal soap
 Chlorhexidine gluconate
 MT 302 Lec- Bacteriology

o Principle: disrupts the microbial cell and precipitate the cell contents.
o 0.5% -0.4% more effective against gram positive than gram negative bacteria and has less
activity against fungi and tubercle bacilli.
o Inactive spores, lipid enveloped viruses but not the nonenveloped viruses like rotaviruses,
and enterovirus.
o Should not come into contact with the eyes, the middle ear, or meninges.
o Binds to the skin and remains active for at least 6 hours.
o pH 5.5- 7.0 for optimum activation.
o Broad spectrum especially against gram negative bacteria
 Hexachlorophene
o Primarily effective against gram positive bacteria.
o Principle: interrupts bacterial electron transport, inhibits membrane-bound enzymes at
low concentrations, and ruptures bacterial membrane at high concentrations.
o Gram positive bacteria: 3% with 15 to 30 seconds
 Chloroxylenol
o A halogen substituted phenolic compound
o Concentration of 0.5-4%
o Principle: cell wall disruption and enzyme inactivation.
o Against gram positive bacteria but not for mycobacterium tuberculosis, fungi, viruses,
and gram bacteria.
o Unaffected by organic substances but neutralized by surfactants and polyethylene glycol.
 Triclosan
o A diphenyl ether that disrupts the cell wall
o Has good activity against gram positive bacteria, gram negative bacteria, and viruses.
o Not affected by organic compounds but affected by pH and surfactant and emollient and
formulation.
o Category I for safety and category III for effectiveness (short used).
o Category III for long term and repeated used.
 Heavy Metals
o Slowly bactericidal and bacteriostatic.
o Examples:
 Mercuric chloride
 Silver nitrite (1% of eyedrop solution)
 Prophylactic treatment to Neisseria gonorrhea in newborn (conjunctivitis)
 Ethylene Oxide
o Gas most commonly used for sterilization.
o 50 to 700 mg of ethylene oxide per liter of chambers at 55 o C to 60 o C for 2 hours.
o Humidity of 30% for optimal destruction of spores.
o Principle. Alkylation of nucleic acid in the spores and vegetative cell.
o Used in hospital materials that cannot withstand steam sterilization.
Handwashing

 Is a good way of antiseptic procedure

 Not just on soap but the process of mechanical rubbing is the one that will activate or make the
soap effective.
 MT 302 Lec- Bacteriology

o Palm to palm
o Between fingers
o Back of hands
o Base of thumbs
o Back of fingers
o Fingernails
o Wrists
o Rinse and wipe dry

Waterless handrubs

 Use of alcohol or sanitizer

 Do if there is no visible dirt in hand.