Microbiology DSA Flashcards
Gram Staining
- Separates organisms into Gram Pos (Blue, absorb Crystal Violet) or Gran Negative (Red, absorb Safranin). Also allows for the shape of the organism to be observed
Steps:
1) Smear the substance to be stained, onto a slide and then heat to fix the bacteria on the slide
2) Pour CRYSTAL VIOLET stain (Blue dye) and wait 60 Secs
3) WASH OFF with water and flood with IODINE Solution. Wait 60 Secs
4) WASH OFF with water and then “DECOLORIZE” with 95% ALCOHOL
5) COUNTER-STAIN with SAFRANIN (Red dye). Wait 30 Secs and WASH OFF with water
Gram Positive Cell Wall
2 Layers:
1) Inner Cytoplasmic membrane (Phospholipid Bilayer)
2) Outer THICK PEPTIDOGLYCAN Layer (60-100% Peptidoglycan)
- LOW Lipid Content (No Cholesterol or other Sterols)
- NO ENDOTOXIN
- NO Periplasmic space
- NO Porin Channel
- VULNERABLE to LYSOZYME and PENICILLIN attacks
- Outer Layer also contains TEICHOIC ACID!!!!
- TRANSPEPTIDASE (Penicillin Binding Protein) catalyzes the formation of the Cross-Linked Peptidoglycan!!!
- PENICILLIN binds to and INHIBITS this enzyme!!!!!
Gram Negative Cell Wall
3 Layers:
1) Inner Cytoplasmic Membrane (Phospholipid Bilayer)
2) THIN PEPTIDOGLYCAN Layer (5 - 10%)
- Contains MUREIN Proteins (Binds 2nd and 3rd layer together)
3) Outer Membrane with LIPOPOLYSACCHARIDE (LPS)
- Outer Part: O Specific Side Chain
- Middel Part: Core Polysaccharide (Water Soluble)
- Inner Part: LIPID A (Endotoxin)
- Can cause Fever, Diarrhea, and possible Fatal Endotoxic Shock
- HIGH Lipid Content
- Has ENDOTOXIN LPS- (LIPID A)
- Has Periplasmic Space
- Has Porin Channel
- RESISTANT to LYSOZYME and PENICILLIN attacks
- NO TEICHOIC ACID!!!!!!
- The Outer Lipid containing cell membrane is partially dissolved by the alcohol during Gram Staining which allows for the Gram Negative Bacteria to pick up the Safranin
Bacterial Morphology
1) COCCI: Spherical
2) BACILLI: Rods
3) SPIRAL FORMS: Comma- shaped, S Shaped, or Spiral-shaped
4) PLEOMORPHIC: lacking a distinct shape like Jello
- PAIRS: Diplo
- CLUSTERS
- STRIPS
- With FLAGELLA
Gram Positive Bacteria Coccus Shaped
1) Streptococcus (Strips)
2) Enterococcus (Strips)
3) Staphylococcus (Clusters)
Gram Positive Bacteria Bacillus Shaped that FORM SPORES
1) Bacillus
2) Clostridium
Gram Positive Bacteria Bacillus Shaped NO SPORES
1) Corynebacterium
2) Listeria
Gram Negative Bacteria Coccus
1) Neisseria (Diplococci)
2) Moraxella (Diplococci)
Gram Negative Bacteria SPIRAL- Shaped
1) SPIROCHETES
- Treponema pallidum (SYPHILIS)
- Borellia
- Leptospira
Gram Negative Bacteria Bacillus
- Almost all Gram Negative Bacteria are Bacilli shaped or Pleomorphic
Pleomorphic:
1) Chlamydia
2) RIckettsiae
Bacteria Exceptions for Shape
1) Mycobacteria
- Weakly Gram Positive
- Stain better with ACID- FAST STAINING!!!!!!!!
- Cause TUBERCULOSIS and LEPROSY!!!!!!
2) Spirochetes
- Have Gram Negative cell wall but are too SMALL to be seen with light microscope
- Visualized with a DARKFIELD MICROSCOPE
- Have THIN Peptidoglycan and LPS membrane
- Surrounded by ADDITIONAL PHOSPHOLIPID-RICH Outer MEMBRANE with few exposed proteins
- This causes Spirochetes to high from Immune Recognition (STEALTH)
- PERIPLASMIC FLAGELLA (Run sideways under outer membrane sheath)
3) Mycoplasma
- Do NOT have CELL WALL
- Only have a simple cell membrane, so neither Gram Pos or Gram Neg
Exotoxin
- PROTEINS that are SECRETED by both Gram Positive and Gram Negative bacteria
- Released by most of the Gram positive
Gram Negative Bacteria:
1) Vibrio cholera
2) Escherichia coli
Diseases caused by Exotoxin:
1) Anthrax
2) Botulism
3) Tetanus
4) Cholera
Exotoxin (NEUROTOXIN)
- Act on the nerves or motor endplates to cause Paralysis
Examples:
1) Tetanis toxin
2) Botulinum toxin
Exotoxin (Enterotoxins)
- Act on the Gastrointestinal tract to cause Diarrhea
- Inhibit NaCl resorption
- Activate NaCl secretion
- Kill Intestinal Epithelial Cells
- CAUSE DIARRHEA
Disease Manifestations:
1) INFECTIOUS DIARRHEA:
- Bacteria colonize and bind to the GI tract, continuously releasing Exotoxin. Diarrhea continues until bacteria destroyed
- Examples:
1) Escherichia coli
2) Campylobacter jejuni
3) Shigella dysenteriae
2) FOOD POISONING:
- Bacteria grow in food and release Enterotoxin in the food. Results in Diarrhea and vomiting
- Examples:
1) Bacillus cereus
2) Staphylococcus aureus
Exotoxin (Pyrogenic Exotoxins)
- Stimulate the release of Cytokines and cause RASH, FEVER, and TOXIC SHOCK Syndrome
- Examples:
1) Staphylococcus aureus
2) Streptococcus pyogenes
Exotoxin (Tissue Invasive Exotoxins)
- Allow bacteria to destroy and tunnel through tissues. Include enzymes that Destroy DNA, Collagen, Fibrin, NAD, RBC, and WBC
Endotoxin
- LIPID A is the best Example of an Endotoxin
- It is a piece of the Outer Membrane Lipopolysaccharide (LPS) of Gram Negative bacteria
- Lipid A is very toxic and is released when the bacterial cell undergoes LYSIS (Destruction)
** Sometimes treating the patient who has GRAM NEGATIVE infection with Antibiotics can WORSEN the patient’s condition because all the bacteria are Lysed, RELEASING LARGE AMOUNT OF ENDOTOXIN!!!!!!!!
- ** Endotoxin is a NORMAL PART of the OUTER MEMBRANE that is shed during LYSIS of the bacteria!!!!!!!
- It is NOT a PROTEIN SECRETED
Virulence Factors
Virulence: the DEGREE of PATHOGENICITY!!!
- Virulence depends on the presence of certain structures and on bacterial Exotoxins and Endotoxins
- Flagella
- Pili
- Capsules
- Endospores
- Biofilms
- Facultative Intracellular Organisms
Flagella
- Protein filaments that extend like long tails from the cell membrane of certain bacteria
- Move the bacteria around
- Flagellum is AFFIXED to the bacteria by a BASAL BODY
- Basal Body spans through the entire cell wall
- Basal body is what SPINS the FLAGELLA
- Single polar Flagellum:
- Vibrio Cholera
- Peritrichous flagella (Many flagella around cell)
- Excherichia coli
- Proteus mirabilis
- PERIPLASMIC FLAGELLA:
- Spirochetes have AXIAL FLAGELLA which run along their unique outer membrane sheath
Pili
- Straight filaments arising from the bacterial cell wall, making bacteria look like a porcupine
- Pili are much shorter than flagella and DO NOT MOVE!!!
- Pili serve as ADHERENCE FACTORS (Adhesins) which are vital for a bacterias ability to cause disease
- Neisseria gonorrhea has pili that allow it to bind to cervical cells and buccal cells to cause gonorrhea
- Escherichia coli and Campylobacter jejune cannot cause diarrhea without their Adhesions to bid to the Interstinal epithelium
- Bordetella pertussis uses its Adhesions to bind to ciliated respiratory cells and cause WHOOPING COUGH!!!
Capsules
- Protective walls that surround the cell membranes of bacteria
- Composed of SIMPLE SUGAR residues
- Bacteria secrete these sugar moieties, which coat their outer wall
- Bacillus anthracis has a Capsule made up of AMINO ACIDS
- Capsules enable bacteria to be MORE VIRULENT because Macrophages and Neutrophils are UNABLE to PHAGOCYTIZE the encapsulated bacteria
- Encapsulated bacteria appear SMOOTH
- Non encapsulated bacteria appear ROUGH
- The process of Ab binding to the capsule is called OPSONIZATION
- Once these Ab have bound to the capsule, Macrophages and Neutrophils can bind to the Fc portion of the Ab and kill the bacteria
Important tests for Capsules
1) India Ink Stain:
- Capsule appears transparent halo around the cell (because stain is not taken up by capsule)
- Identify fungus Cryptococcus
2) Quellung Reaction:
- The bacteria are mixed with Antibodies that bin to the Capsule
- When these Ab bind, the capsule SWELLS WITH WATER, and this can be visualized microscopically!!!
Endospores
- Formed by only 2 Gram Positive bacteria:
1) Bacillus (aerobic)
2) Clostridium (anaerobic) - Endospores are metabolically dormant forms of bacteria that are resistant to heat (boiling), cold, dying, and chemical agents
a) Cell Membrane
b) Thick Peptidoglycan mesh
c) Another Cell Membrane
d) Wall of Keratin-like Protein
e) Outer layer called EXOSPORIUM - Spores form when there is a shortage of needed nutrients and can lie dormant for years!!!!!!!!!
- Autoclave is used to kill the bacteria that form spores
- 121 degrees Celsius for 15 mins, with steam under pressure
- Autoclave is used to kill the bacteria that form spores