-
Selective Mediums
- Either inhibits the growth of a certain group of microbes or supports the growth of very few microbes
- Antibiotics
- Salts
- Toxic Dyes
- Limit carbon/energy source
- Either Gram + OR Gram -
-
Differential Mediums
- Distinguish closely related organisms from one another (usually biochemical abilities)
- Typically a visual difference (pH indicator)
-
Staphylococcus 110
- A type of selective medium
- Contains NaCL and mannitol
- Lacks phenol red
- Favors colony pigmentation by different strains of S. aureus
- No color change as mannitol is fermented
-
Blood Agar Plate
A type of differential medium
-
Alpha Hemolysis
- A partial breakdown of hemoglobin to methemoglobin
- Red to green
-
Beta Hemolysis
Capable of completely hydrolyzing hemoglobin
-
Gamma Hemolysis
Possess no hemolytic actions whatsoever
-
MacConkey Agar (MAC)
- Indicator: Neutral red, pH
- Inhibitor: Crystal violet and bile salts are toxic to gram +
- Results:
- -Gram += inhibited growth
- -Gram - fermenting lactose= pink colonies
- -Gram - not fermenting lactose= colorless colonies
-
Eosin Methylene Blue Agar (EMB)
- Indicator: Methylene blue and eosin Y
- Inhibitor: Methylene blue is toxic to gram +
- Results:
- -Gram += inhibited growth
- -Gram - fermenting lactose= pink or purple colonies with a metallic green sheen
- -Gram - not fermenting lactose= colorless colonies
-
Salmonella Shigella Agar (SS)
- Indicator: Neutral red for lactose fermentation, FeS for H2S production
- Inhibitor: Sodium citrate and brilliant green dye are toxic to most bacteria except these
- Results:
- -Gram += inhibited growth
- -Gram - fermenting lactose= pink colonies; if H2S is produced, the center of the colony will be black
- -Gram - not fermenting lactose= colorless colonies
-
Mannitol Salt Agar (MSA)
- Indicator: Phenol red, pH
- Inhibitor: 7.5% NaCl
- Results:
- -Gram + staphylococci fermenting mannitol= yellow media, neutral pH
- -Gram + staphylococci not fermenting mannitol= red media, basic pH
- -Gram + streptococci= inhibited growth
- -Gram -= inhibited growth
- NaCl inhibits other organisms than staphylococci
-
Use a drop of isopropanol on lens paper
How to Remove Immersion Oil
-
Oil Immersion
- Ensures most light will travel straight into the objective lens
- Prevents light from being lost to the surrounding air
-
Simple Staining
- Two types of dye- acidic and basic
- Used to see the morphology and shape of bacteria
-
Positive Simple Stain
Colors the cell, leaving the background colorless
-
Negative Simple Stains
Cell remains colorless, the background retains the stain
-
Gram Negative
- Any organism that takes up the red safranin in gram staining
- Red
- Have a higher lipid content in the cell wall
- Lipids are dissolved by the alcohol causing the crystal violet to leak from the cell
-
Gram Positive
- Any organism that retains the primary dye in gram staining
- Purple
- Have a thick layer of peptidoglycan
- Pores in the cell wall are closed and prevent the loss of the crystal violet
-
Differential Staining
A method to distinguish between different types of bacteria
-
Bacillus, Clostridium and Sporosarcina
Common species that form endospores
-
Defined Media
- Composed of designated amounts of specific compounds
- Exact contents are known
- Amounts are known
-
Complex Media
- Contain nutrient-rich substances and precise chemical constituents are unknown
- Amounts of chemicals are not known
- Yeast extract, peptone, and tryptone are some substances are used
-
Different nutrient needs
Different metabolic needs
Why can't all bacteria be cultured?
-
Streak Plate
- Useful in isolating colonies
- Can be made from broth and/or plate cultures
-
Spread Plate
- Useful in quantifying bacteria
- Also isolate colonies (if proper dilution is used)
- Must be done with a liquid culture
-
Zone of Inhibition
- A function of molecular structure weight, charge, solubility, and diffusion within the medium
- Indicates some chemical activity occurred in vitro
-
Spoilage
When microbes grow within or on the surface of foods
-
Pathogenic Spoilage
A potential major health hazard
-
Cured
Smoked
Broiled
Types of fermenting
-
Microbial growth is slowed
Metabolism is delayed due to the slower responses of bacterial enzymes
Why store food at low temperatures
-
Thermophiles and Hyperthermophiles
- Live in very hot conditions (above 45C)
- Compost heaps, hot springs
-
Mesophiles
- Live in conditions like body temperature (~37C)
- Most human pathogens
-
Psychrophiles
- Live in cooler climates
- Refrigerators (4C)
- Causes spoilage of refrigerated foods
-
Adding salt, sugar, and/or some spice
Can lead to bacterial cell destruction
What changes the osmotic conditions of food environments?
-
Halophiles
Live in high salt conditions
-
Saccrophiles
Live in high sugar concentrations
-
Neutrophiles
Live in neutral pH environments
-
Acidophiles
Live in more acidic pH's
-
Alkalophiles
- Live in higher pH environments
- Degrade proteins for their energy
- Produce amines and ammonia
-
Refrigeration
pH changes
Addition of salt, sugar, vinegar, or some spices
What can prevent food spoilage?
-
They are born
Bacteria begin the colonization process
Babies are sterile until...
-
Commensal Organisms
- Live with humans in a harmonious relationship
- Normal flora
-
Saprophytes
Live off the nutrients of dead cells and tissues
-
Metabolic end products which are absorbed by their human host
Vitamins
Some bacteria produce...
-
Environment
Body chemistry
Age
Hormones
Body secretions
Diet
Climate
Physical activities
Antibacterial therapy
Specific growth environments
What changes the structure of the normal flora community in a host?
-
Staphylococcus
- Normal flora for the skins
- Deadly pathogen if introduced in the bloodstream
-
Skin
- Normal flora:
- Staphylococcus
- Corynebacterium
- Proprionibacterium
- Streptococcus
-
Throat
- Normal flora:
- Streptococcus
- Corynebacterium
- Neisseria
- Branhamella
-
Large Intestine
- Normal flora:
- Escherichia
- Bacteriodes
- Lactobacillus
- Candida
- Clostridium
- Enterococcus
-
Urethral/Anal Openings
- Normal flora:
- Escherichia
- Lactobacillus
- Enterococcus
- Bacteriodes
- Streptococcus
-
Vaginal Openings
- Normal flora:
- Lactobacillus
- Candida
- Stretococcus
-
Skin
- Dry environment
- Acidic pH
- Inhospitable for many microbes
- Microflora are gram-positive
- Anaerobic bacteria living in pores
-
Propionibacterium acnes
- Metabolizes sebum
- Releases fatty acids
- Enhance anti-microbial environment provided by the skin
-
Mannitol Salt Agar (MSA)
Staphylococcus Medium 110 (SM110)
Two medias used for primary isolation on skin
-
Staph and strep
Gram stains help differentiate between what two bacterias?
-
Catalase Test
- Checks for the presence of the enzyme catalyse
- Splits hydrogen peroxide into hydrogen and water
- Bubbles= positive
- Lack of bubbles= negative
-
Catalase Positive
- Gram positive cocci
- Either micrococci or staphylococcus
-
Catalase Negative
- Gram positive cocci
- Streptococcus
-
Beta-Hemolytic Streptococci
- Belong to Lancefield groups A, B, and D
- Most important pathogens
-
Hemolysins
- Uses blood agar
- BAP distinguishes between 3 different types of hemolysis
-
Alpha Hemolysis
- Greenish zone around the colony
- Due to partial breakdown of red blood cells
- Most bacillus species
-
Beta Hemolysis
- Clear around the colonies
- Complete lysis of the red blood cells
- Strep pyogenes, Staph aureus, and all gram negative enteric bacteria
-
Gamma Hemolysis
- No hemolysis
- Staph epidermis and Strep faecalis
-
Blood degeneration
Over incubation in the hemolysins' test causes
-
Coagulase Test
- Confirms Staphylococcus aureus
- 97% of strains are positive
- Others are negative
- Test only valid for gram-positive
- Some gram-negative rods cause false-positive reations
-
Positive Coagulation
- A loose clot
- Solid immovable clot
-
Negative Coagulation
Slanted agar
-
Hydrolases
Split complex organic compounds into smaller units in the presence of water
-
Amylase and Gelatinase Hydrolysis Test
Identify bacteria
-
Amylase
- Bacteria that hydrolyze starch produce..
- Clear
-
Molecules of maltose, glucose, and dextrin
The breakdown of starch produces...
-
Dyes starch blue
Indicates a lack of starch hydrolysis
Iodine and Starch
-
Gelatinase
- Gelatin is a protein that can be broken down by the enzyme..
- Indicated by the liquid in the tube
-
No Starch Hydrolysis
Medium has a clear zone
-
Mannitol Salt Agar (MSA)
Staphylococcus Medium 110 (SM110)
Two medias used for primary isolation in the nose
-
-
Blood Agar Plates (BAP)
Enriched media loaded with vitamins and nutrients
Allows detection of alpha and beta hemolytic streptococci
What type of agar is used for the throat?
-
CAMP Test
Help in the identification of Streptococci that produce beta hemolysis
What type of test is designed for the throat?
-
Optochin Susceptibility
Differentiates alpha-hemolytic viridans group streptococci from the pneumococci
-
Camp Factor
An enlarged arrowhead-shaped hemolytic zone at the juncture where the unknown meets the S. aureus means it is positive
-
Cultivating fecal samples of selective and differential media which contain lactose
Enteric bacteria may be isolated by...
-
MacConkey agar
Levine eosin methylene blue (EMB)
Two types of agar used for normal flora of the intestinal tract
-
MacConkey Agar
Contains bile salts to inhibit non-enteric bacteria and two dyes, neutral red and crystal violet, which are taken up by lactose-fermenting bacteria
-
EMB Agar
Inhibitory to gram-positive bacteria
-
Lactose-Fermenting Bacteria on MAC
- Appears red
- E. coli is brick red
- Enterobacter, Klebsiella and other lactose fermenters are pink to red with a mucoid texture
-
Gram Stains in Intestinal Tract
Isolates to confirm the presence of gram-negative rods
-
Results of EMB of Intestinal Tract
- E. coli- blue-black with a green metallic sheen in reflected light
- Enterobacter aerogenes- convex and mucoid with dark centers and creamy pink borders
-
Triple Sugar Iron (TSI) Agar
- Valuable medium for the further differentiation of enteric bacteria
- Contains three carbohydrates: glucose, lactose, and sucrose
- pH indicator phenol red
- Anaerobic conditions
- Contains iron ions and sodium thiosulfate
-
Gas has been produced
Large amounts of gas may push the medium up the tube
Cracks in the agar of TSI means..
-
There is H2S reacting with iron making iron sulfide
If the TSI medium is black, that means...
-
TSI medium
Bacteria isolated on MAC and EMB plates will be differentiated by their reactions in...
-
Indole production
Methyl red test
Voges-Proskauer test
Citrate test
IMViC Series
-
Indole Test
- Amino acids are produced as a result of the hydrolytic degradation of proteins, peptones, and peptides
- Tryptophan
-
Methyl Red Test
- Depends on an organism's ability to ferment glucose to acid
- Red in acid solutions
- Yellow in alkaline or neutral solutions
-
Voges-Proskauer Test
- Depends on the digestion of glucose to acetylmethylcarbinol
- If present it will form a red color
-
Citrate Test
- Based on the ability of certain bacteria to utilize a salt of a citric acid as a sole carbon source in growth
- Indicator bromthymol blue becomes deep blue when pH of medium rises
- If utilization does not occur, then appears green
-
Indole Production Results
- Positive- presence of a red surface layer
- Negative- no production
-
Methyl Red Results
- Positive- acid is produced- red
- Negative- yellow
-
Voges-Proskauer Results
- Positive- red
- Negative- lack of color change
-
Citrate Test Results
- Positive- ability to utilize citrate- blue
- Negative- green
-
E. coil, followed by Pseudomonas aeruginosa, and Staphylococcus aureus and s. epidermis
Common cause of UTIs
-
Inflammation of the bladder (cystitis)
Inflammation of one or both kidneys (pyelonephritis)
Two major infections of the urinary tract
-
Blood Agar Plates (BAP)
Eosin-Methylene Blue (EMB)
Two types of agar used for primary detection in urinary tract
-
30-300
Fewer than means could not be statistically validated
More than means there are colonies touching
Range of countable colonies
-
Dilution
Transfer of a known volume of a sample into a known volume of diluent
-
volume of sample added
volume of sample added + volume of diluent
Dilution=
-
Dilution 1 x dilution 2 x dilution 3...
Total dilution=
-
1
total dilution
Dilution Factor=
-
# of colonies x dilution factor on a countable plate
= # of bacteria in the original sample
-
Compare to chart
If positive, circle number
How do you score an enterotube test?
-
Positive- indole and methyl red
Negative- citrate and voges-proskauer
What tests are E. coli positive and negative in IMViC?
-
Citrate- bromethymol blue
Methyl red- methyl red
What is the indicator for citrate and methyl red test?
-
Fermentation
Gas production
H2S production
What are 3 general outcomes of TSI slant?
-
Nosocomials
Hospital acquired infections
-
MAC- methylene blue
EMB- neutral red
What are the indicators in MAC and EMB agar?
-
Positive
Bacteria that grows on the outside of the body is...
-
Negative
Bacteria that grows on the inside of the body is...
-
E. auregenus and E. coli
What are the two bacteria used in IMViC?
-
Diplo-
Pair of cocci or bacilli
-
Strepto-
chain of three or more cocci or bacilli
-
Staphylo-
Cluster of cocci
-
Tetrad
Packet of four cocci in a square
-
Sarcina
Packet of eight cocci in a cube
-
The inside
What part of your body is sterile?
-
Enterococci and E. coli
What two types of bacteria are used in water?
-
Protein and starch hydrolysis
What are two types of hydrolysis reactions?
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