Introduction to Microbiology

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Introduction to Microbiology MIC241(Level 4)
Course Director Dr. Dalia Mohsen
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Lecture 1Microbial World And You
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What is Microbiology?Microbiology is the study
of microorganisms.

• Micro - too small to be seen with the naked eye
  (cannot seen by naked eye).
• Bio - life
• ology - study of

What are Microorganisms? Also called as Microbes
are minute living things that individually are
usually too small to be seen with the unaided
eye. An organism that can be seen only with
the aid of a microscope.
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Characteristics of cell

• Maintain structure by taking up chemicals and
  energy from the environment
• Respond to stimuli in the external environment
• Reproduce and pass on their organization to
  their offspring
• Evolve and adapt to the environment

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Study of various groups of Microorganisms

• 1. Bacteria
• 2. Protozoans
• 3. Algae
• 4. Parasites
• 5. Yeasts and Molds
• (Fungi)
• 6. Viruses

• Bacteriology
• Protozoology
• Phycology
• Parasitology
• Mycology
• Virology

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Microorganisms - what comes to mind?

• Diseases
• Infections
• Epidemics
• Food Spoilage
• Only 1 of all known bacteria cause human
  diseases
• About 4 of all known bacteria cause plant
  diseases
• 95 of known bacteria are non-pathogens

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• Microorganisms are found everywhere in the world,
  in all living things, plants and animal, on and
  inside your body, than there are cells that make
  up your entire body.
• Microorganisms can live in the air, on land, and
  in fresh or salt water environments.
• Some of them are pathogens, can be harmful and
  causes diseases, but majority of microorganisms
  beneficial for us.

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• Escherichia coli
• Bacillus anthracis
• Salmonella enteritis
• Streptococcus pyogenes
• Streptococcus lactis
• Streptococcus faecalis
• Erlichia canis
• Campylobacter jejuni
• Helicobacter pylori
• Enterobacter aerogenes

• Staphylococcus aureus
• Staphylococcus epidermidis
• Streptococcus pneumoniae
• Vibrio cholerae
• Rhodospirillium rubrum
• Bacillus subtilis
• Micrococcus luteus

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Types of Microorganisms

1. Bacteria are unicellular organisms. Because they
   have no nucleus, the cells are described as
   prokaryotic.
2. Archaea (Archae Bacteria) have prokaryotic
   cells lack peptidoglycan in their cell walls.
   Archaea include methanogens, halophiles, and
   extreme thermophiles.

extreme thermophiles
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• 3. Fungi (mushroom, molds, and yeasts) have
  eukaryotic cells (with a true nucleus). Most
  fungi are multicellular. Fungi obtain nutrients
  by absorbing organic material from their
  environment.
• 4. Protozoa are unicellular eukaryotes. Protozoa
  obtain nourishment by absorption or ingestion
  through specialized structures.

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• 5. Algae are unicellular or multicellular
  eukaryotes that obtain nourishment by
  photosynthesis. Algae produce oxygen and
  carbohydrates used by other organisms.
• 6. Viruses are non cellular entities that are
  parasites of cells. Viruses consist of a nucleic
  acid core (DNA or RNA) surrounded by a protein
  coat

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Lecture 2

• Functional Anatomy of Prokaryotic and Eukaryotic
  Cells

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Cell is the structural and functional unit of an
organism

• All living cells are classified into Prokaryotic
  and Eukaryotic Cells, based on their structural
  and functional characteristics.
• Prokaryote comes from the Greek words for
  pre-nucleus.
• Eukaryote comes from the Greek words for true
  nucleus.

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• Organisms with
• PROKARYOTIC CELLS Bacteria and Archaea.
• EUKARYOTIC CELLS fungi, protozoa, algae, plants
  and animals.
• Viruses -Non-cellular elements that do not fit
  into any organizational scheme of living cells.
  (will be discussed later)

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PROKARYOTIC CELLS
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Bacterial Morphology

• Bacteria are unicellular.
• Most bacteria are 0.2 um in diameter and 2-8 um
  in length.
• Most bacteria are monomorphic maintain a single
  shape.
• and few are pleomorphic they can have many
  shapes. Ex Rhizobium and Corynebacterium.

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• Basic shapes of bacteria
• COCCI
• Cocci may be oval, elongated, or flattened on one
  side.
• Cocci may remain attached after cell division.
  These group characteristics are often used to
  help identify certain cocci.

Cocci that remain in pairs after dividing are
called diplococci.
Cocci that remain in chains after dividing are
called streptococci.
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Cocci that divide in two planes and remain in
groups of four are called tetrads.
Cocci that divide in three planes and remain in
groups or cube like groups of eight are called
sarcinae.
Cocci that divide in multiple planes and form
grape like clusters or sheets are called
staphylococci.
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BACILLI

• Bacillus means rod shaped.
• Bacilli only divide across their short axis, so
  there are fewer groups.
•  

Most bacilli appear as single rods. Diplobacilli
appear in pairs after division.
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SPIRAL BACTERIA
Spiral bacteria have one or more twists.
Vibrios look like curved rods
Spirilla have a helical shape and fairly rigid
bodies.
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• Spirochetes have a helical shape and flexible
  bodies.
• Spirochetes move by means of axial filaments,
  which look like flagella contained beneath a
  flexible external sheath.

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Unusual shapes
Stella are star-shaped.
Haloarcula, a genus of halophilic archaea, are
rectangular.
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Streptobacilli appear in chains after division.
Some bacilli are so short and fat that they look
like cocci and are referred to as Coccobacilli.
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STRUCTURE OF A PROKARYOTIC CELL
virulent factor
movement
attachment
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STRUCTURE OF A PROKARYOTIC CELL

• The structure is described according to the
  following organization
• Structures, external to cell wall
• Structure of cell wall
• Structures, internal to cell wall

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Structures External To The Cell Wall

• Glycocalyx
• Flagella
• Axial filaments
• Fimbriae
• Pili

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GLYCOCALYX(sugar coating)

• The glycocalyx (capsule, slime layer, or
  extra cellular polysaccharide) is a gelatinous
  polymer.
• A capsule is neatly organized
• A slime layer is unorganized loose
• External to cell wall, composed of
  polysaccharide, polypeptide covering or both.
• The presence of a capsule can be determined by
  negative staining.

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• Capsules are important in contributing to the
  virulence of the bacteria.
• Protect bacteria by preventing phagocytosis.
• Allows the bacteria to adhere and colonize.
• Important components of biofilm
• protects cell
• Facilitates communication among them
• Enable to survive by attaching to various
  surfaces
• Protects cell against dehydration
• Inhibit the movement of nutrients out of the
  cell.

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• Capsulated bacteria
• Streptococcus pneumoniae
• Klebsiella pneumoniae
• Haemophilus influenzae
• Bacillus anthracis
• Streptococcus mutans
• Yersinia pestis

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Streptococcus pneumoniae (in vivo)
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K. pneumoniae
Haemophilus influenzae
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FLAGELLA

• Long filamentous appendages consisting of a
  filament, hook, and basal body
• Made of chains of protein (flagellin)
• Attached to a protein hook
• Anchored to the wall and membrane by the basal
  body
• Semi rigid, helical structure that moves the cell
  by rotating from the basal body.

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Flagella are anchored by pairs of rings
associated with the plasma membrane and cell
wall. Gram positive bacteria have only the inner
pair of rings
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Flagella Arrangement
Peritrichous distributed over the entire cell
Monotrichous single flagellum at one pole
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Amphitrichous flagella at both poles of cells
Lophotrichous a tuft of flagella coming from
one pole
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Motility

• Rotate flagella to run or tumble
• Move toward or away from stimuli (taxis)
• The stimuli include chemicals like oxygen,
  ribose, galactose Chemotaxis.
• Stimuli can be light Phototaxis.

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• Flagellar (H) protein functions as an antigen.
• Flagella proteins are H - antigens - useful in
  distinguishing the variants within the species of
  gram-negative bacteria.
• Example 50 different H antigen for E. coli are
  identified.
• E. coli O157H7 associated with food borne
  epidemics.

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AXIAL FILAMENTS

• Also known as Endoflagella are bundles of
  fibrils that arise at the ends of the cell
  beneath an outer sheath and spiral around the
  cell.

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• Spiral cells that move by means of an axial
  filament are called spirochetes.
• Axial filaments are similar to flagella, except
  that they wrap around the cell.
• Anchored at one end of a cell
• Rotation causes cell the movement of the outer
  sheath that propels the spirochetes in a spiral
  motion.

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FIMBRIAE AND PILI

• Fimbriae and pili are short, straight, thin, hair
  like appendages.

• Made up of protein called Pilin.
• Arranged helically around a central core.

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• Fimbriae
• Occur at poles or evenly distributed.
• Number can vary from few to several hundreds
• Allow attachment to surfaces and adhere to each
  other
• Pili
• Longer than Fimbriae
• Only one or two per cell
• are used to transfer DNA from one cell to another
  by Conjugation (sex Pili).
• Involved in motility called twitching motility
  short jerky intermittent movements, seen in
  Neisseria gonorrhoeae.
• Other type of motility is gliding motility
  smooth gliding movement of mycobacterium.

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Structure of Cell Wall

• Is a complex, semi rigid structure responsible
  for the shape of the cell.
• Surrounds the underlying, fragile plasma
  membrane.

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• Functions
• Prevents osmotic lysis
• Keep or protect the cell shape
• Point of anchorage for flagella
• In some species it has the ability to cause
  disease and is the site of action for some
  antibiotics.

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Composition and Characteristics

• PEPTIDOGLYCAN - Main component of bacterial cell
  wall (also known as murein) - a polymer
  consisting of disaccharideN-acetyl glucoseamine
  (NAG) N-acetyl muramic acid (NAM) linked by
  polypeptides chains.

N-acetyl glucosamine (NAG) and N-acetyl muramic
acid (NAM) joined as in peptidoglycan
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• Alternating NAM and NAG molecules form a
  carbohydrate backbone (the glycan portion).
• Rows of NAG and NAM are linked by polypeptides
  (the peptido - portion).
• The structure of the polypeptide cross-bridges
  may vary but they always have a tetra peptide
  side chain, which consists of 4 amino acids
  attached to NAMs. The amino acids occur in
  alternating D and L forms.

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Gram positive cell wall

• consist of many layers of peptidoglycan and also
  contain teichoic acids.
• Teichoic acids may
• bind and regulate movement of cations into and
  out of the cell
• prevent extensive wall breakdown and possible
  cell lysis during cell growth
• provide much of the cell wall's antigenicity

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Gram negative cell wall

• Have a lipopolysaccharide-lipoprotein-phospholipi
  d outer membrane surrounding a thin (sometimes a
  single) peptidoglycan layer.
• Gram-negative cell walls have no teichoic acids.

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• Forms the periplasm between the outer membrane
  and the plasma membrane.
• Protection from phagocytes, complement,
  antibiotics like penicillin, lysozyme, and other
  chemicals. .
• O polysaccharide antigen, e.g., E. coli O157H7.
• Lipid A is an endotoxin causes fever and shock
• Porins (proteins) form channels through membrane

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Mycobacteria Cell Wall

• Like Mycobacterium tuberculosis, Mycobacterium
  leprae
• Contains Mycolic acid layer (waxy layer) instead
  of Peptidoglycan layer

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Atypical Cell Walls

• Mycoplasmas
• Smallest known bacteria
• Lack cell walls
• Sterols in plasma membrane protect them from
  lysis.
• Archaea
• Wall-less, or
• Walls of pseudomurein (lack NAM and D amino acids)

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Damage to Cell Walls

• Lysozyme digests disaccharide in peptidoglycan.
• In the presence of lysozyme, gram-positive cell
  walls are destroyed, and the remaining cellular
  contents are referred to as a protoplast.
• In the presence of lysozyme (after disruption of
  the outer membrane), gram-negative cell walls are
  not completely destroyed, and the remaining
  cellular contents are referred to as
  spheroplasts.

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• Protoplasts and spheroplasts are susceptible to
  osmotic lysis.
• Protoplasts and spheroplasts are capable to
  re-gain their cell wall.
• Penicillin inhibits peptide bridges in
  peptidoglycan.
• Proteus and some other genera can lose their cell
  walls spontaneously or in response to penicillin
  and swell into L forms. L forms can live and
  divide and/or return to the normal walled state.
• L-forms (Mycoplasma ) are natural wall-less cells
  that swell into irregular shapes.

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Structures Internal To The Cell Wall

• Plasma membrane
• Cytoplasm
• Nucleoid
• Ribosomes
• Inclusions

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PLASMA MEMBRANE

• The plasma membrane encloses the cytoplasm and is
  a phospholipid bilayer with peripheral and
  integral proteins (the fluid mosaic model).
• The plasma membrane is selectively permeable.

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• Phospholipid bilayer
• Peripheral proteins
• Integral proteins
• Trans-membrane proteins

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RIBOSOMES

• The cytoplasm of a prokaryote contains numerous
  70s ribosomes ribosomes consist of rRNA and
  protein.
• Protein synthesis occurs at ribosomes it can be
  inhibited by certain antibiotics.
• The difference between prokaryotic (70s) and
  eukaryotic (80s) ribosomes allows antibiotics to
  selectively target the prokaryotic ribosomes
  while sparing eukaryotic ribosomes.

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INCLUSIONS

• Phosphate reserves
• Energy reserves
• Ribulose 1,5-diphosphate carboxylase for CO2
  fixation
• Protein covered cylinders
• Iron oxide (destroys H2O2)

• Metachromatic granules (volutin)
• Polysaccharide granules and Lipid inclusions and
  Sulfur granules
• Carboxysomes
• Gas vacuoles
• Magnetosomes

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ENDOSPORES
Endospores are resting structures formed by some
bacteria for survival during adverse
environmental conditions.

• The process of endospore formation is called
  sporulation the return of an endospore to its
  vegetative state is called germination.  Two
  genera that commonly form endospores are Bacillus
  and Clostridium.

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Culturing Microorganisms
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Culturing Microorganisms

• Microbiologists culture microorganisms by
  transferring an Inoculum.
• Inoculum (a sample) from a clinical or
  environmental specimen
• into a medium, a collection of nutrients. Liquid
  media are called broths.
• Microorganisms that grow from an Inoculum are
  called a culture.

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Culture Media

• Culture Medium Nutrient material prepared for
  microbial growth in the laboratory.
• Properties of culture media
• Must contain right nutrients
• Should contain sufficient moisture
• Adjusted pH
• Suitable level of oxygen
• Must be sterile (contain no living microorganism)

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Agar-Agar

• Derived from marine algae
• Agar, a complex polysaccharide
• Useful compound because it is difficult for
  microbes to digest
• It solidifies at temperatures below 40C
• It does not melt below 100C.
• Used as solidifying agent for culture media in
  Petri plates, slants, and deeps
• In order to create solid media in Petri dishes,
  freshly prepared, warm, liquid agar is poured
  directly into the dish and cooled at room
  temperature.
• Slant tubes, or slants are created by pouring
  liquefied agar into test tubes, placing them at
  an angle until the agar solidifies.
• And as a deep if solidified in a vertical tube.

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• Anaerobic Jar

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Anaerobic chamber
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Special culture techniques

• Carbon dioxide incubator
• Grows aerobic bacteria that require CO2
  concentrations, higher or lower than that found
  in the atmosphere.
• Desired CO2 levels is obtained by
• Electronic methods
• Sealed candle jar
• Chemical packets that generate CO2
• Capnophiles organisms that require high CO2
  concentration.

CO2-packet

• Candle jar

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• Mycobacterium leprae is usually grown in
  Armadillos (mammals with a leathery armor shell),
  as they have a body temperature of 34 C
  (93 F), similar to human skin.

Armadillos

• Obligatory intracellular bacteria like rickettsia
  and Chlamydia, like viruses reproduce only in
  living host.

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Selective Media

• Typically contain substances that induce the
  growth of particular microorganisms or inhibit
  the growth of unwanted ones.

Sabouraud agar (acidic pH selective for fungi).
In the picture of the Sabouraud plate - notice
the growth of the mold (fuzzy appearance) and the
absence of bacteria
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Differential Media

• Growth of microbes on differential media results
  in visible differences in the growth of two or
  more organisms.
• Used in conjunction with selective media to
  create a whole class of media called selective
  and differential media.
• Differential Agent - represents a chemical that
  allows you to distinguish one bacteria from
  another.

• Example- Mannitol salt agar (MSA) - supports the
  growth of halophiles.

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Enrichment Media

• Encourages only the growth of a desired microbe,
  which is often missed as they are small in
  number.
• Is usually liquid, and provides nutrients and
  environmental conditions that favors only a
  particular organism.
• Ex - Diarrheal sample contains a few Salmonella
  bacteria and thousands of other bacteria.
• Inoculate Salmonella-containing sample to S-S
  culture medium then incubate at 37oC.
• Only Salmonella will be growing, even if it is
  present in few numbers and while others do not
  grow.

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Rich medium

• Media support the growth of almost of all
  microorganisms like blood agar, chocolate agar

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Obtaining Pure Cultures

• A pure culture contains only one species or
  strain of bacteria
• Suspected pathogens must be isolated from the
  normal microbiota in pure culture
• A colony is a population of cells arising from a
  single cell or spore or from a group of attached
  cells
• A cell is often called a colony-forming unit (CFU)

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Streak Plate Method
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Preserving Bacteria Cultures

• Deep-freezing a process in which a pure culture
  of microbes is placed in a suspending liquid and
  quickly frozen at a temperature ranging from -50
  to -95C
• Lyophilization (freeze-drying) - a suspension
  of microbes is quickly frozen (-54 to -72C) and
  dehydrated in high vacuum (sublimation)

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Direct Measurements of Microbial Growth

• Perform serial dilutions of a sample

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Plate Count

• Inoculate Petri plates from serial dilutions

Figure 6.16
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Plate Count

• After incubation, count colonies on plates that
  have 25-250 colonies (CFUs)