Lectures 7, 8 Flashcards
What is the first quickest step to identifying a bacteria?
Look at it under the microscope
What is the difference between prokaryotes and eukaryotes?
Prokaryotes- misty circular chromosomes
No organelles
Peptidoglycan cell walls
Binary fission
Eukaryotes- mostly linear chromosomes, in nuclear membrane
Organelles
Polysaccharide cell walls
Mitosis
L7S4
What are the different bacteria shapes and what are their Latin names?
Spherical= coccus/cocci
Rods= bacillus/bacilli
Long and bendy= vibrios (crescent shaped), spirillas, spirochetes
L7S5
How are pairs, clusters and chains named in Latin with different bacterial shapes?
How are the groupings related to plane of cell division?
Pairs= diplo (diplococci, diplobacilli)
Clusters= staphyl (staphylococci)
Random but stuck together
Chains= strepto (streptococci, streptobacilli)
Chains only divide on length since they don’t divide horizontally
Pots can divide in any plane
L7S6
Study the anatomy of a bacterium on L7S7
Ok
What is the bacterial cell wall?
What are the two types of stain and their colours?
What is the structure of the bacterial cell wall?
Protects cells from mechanical and osmotic forces
Gram stain developed in 1884 (Hans Christian gram)
Gram negative cells are pink
Gram positive cells are purple
The cell wall is threads of repeating carbohydrate (NAG-NAM) glued together with proteins
These sugars and proteins form the compound peptidoglycan
L7S9
Why is the bacterial cell wall cross linked?
It makes the wall durable
NAG-NAM formed outside the cell and brought in then cross linking is done by enzyme peptidase
What are gram positive cells?
5 common ones?
L7S11
Multiple layers of peptidoglycan
Teichoic acids aid in keeping layers stick together and increase - charge in cell wall
Streptococcus pneumoniae Staphylococcus aureus Clostridium botulinum Clostridium difficile Bacillus anthracis
What are gram negative cells?
L7S13
Single layer of peptidoglycan
Second phospholipid membrane outside the peptidoglycan (membrane helps repel some immune system factors, block entry of antibiotics and can contain toxic compounds
Neisseria gonorrhoeae Haemophilus influenzae Helicobacter pylori (stomach ulcers) Escherichia coli O157:H7 (O polysaccharide, H7 is part of flagella, makes toxins that do kidney damage) Vibrio cholerae
What are the 5 types is bacterial staining?
Gram stain Acid fast stain Negative stain Flagella stain Endospore stain
What are bacterial smears?
First step in staining
Mix coming of bacteria with a drop of water or place drop of liquid culture on the slide
Allow to air dry then heat fix (heat melts sugars to the surface of the glass)
Need wax ring to tell front and back slide apart
What is the gram stain process?
L7S17
- Apply crystal violet for 1 minute (highly permeable stain, all cells turn purple)
- Then wash off and apply iodine (mordant) for 1 minute (licks crystal violet in place)
- Wash off then apply alcohol wash (acetone) for decolourization, this results in gram positive stains as purple and gram negative is colourless (exposes thin layer of peptidoglycan which isn’t thick enough to hold crystal violet)
- Wash off quickly and apply safranin (counterstain) for 1 minute (stains all cells and adds pink colour to all cells) then rinse with water
What are some gram staining difficulties?
- Older cultures have weak cell walls (lose CV)
- Older solutions may not work properly (especially iodine doesn’t work as mordant if it’s old which won’t lock crystal violet in cells)
- Decolourization timing is critical (easy to do too much or too little, too high makes positives become negatives and negatives will stay as negatives, too low makes positives look positive and negatives look negative
- excessive counterstain can displace CV
- some bacteria are gram variable
What is the acid fast stain?
Each cell walls repel gram stains (and antibiotics)
Mycobacterium tuberculosis, mycobacterium leprae
Primary dye is carbol-fuschin (high affinity for waxy mycolic acids)
Decolourization with an acid-alcohol removes dye from most cells
Counter stain with methylene blue
Slide 3
What is the glycocalyx?
What are it’s subgroups capsule and slime layer?
Outside the cell wall
Increases pathogenicity
Composed of sugars & proteins
If you have a glycocalyx you have a better chance at coating yourself from bacteria with an extra coat
(Doesn’t stain very well so try and not have it stained)
Capsule- neatly organized, tight
Slime layer- unorganized and loose/diffuse (makes cells look slimy and loose)
Slide 4
What is negative stain?
Allows visualization of glycocalyx
Nigrosin or Congo red dyes
(Sizing and charge prevent binding to or penetrating bacteria)
Thin film penetration
Cells are not fixed
Additional staining to visualize bacteria
Slide 5-6
What is the flagella?
Used for motility
10-20nm in width
(Maximum resolution for a light microscope is 200nm)
Need to thicken the flagellum to view with a light microscope- leifson stain= crystal violet + a mordant
Slide 7
What are the 4 flagella patterns (plus one outlier pattern)?
Monotrichous- one flagella Amphitrichous- one out each end Lophotrichous- two out each end Peritrichous- multiple flagella scattered Atrichous- no flagella
Slide 8-9
What are endospores?
A dormant daughter cell which is formed inside the main cell
Resists drying out, heat, chemicals, radiation (prevents chemicals from getting in it disinfectant doesn’t destroy endospores)
Is not alive, all cellular processes are halted
Bacillus and clostridium
Study the formation of endospore on slide 11
Okay
What is an endospore stain?
Use moist heat to stain the endospore wall (hard to stain since impermeable to stain and antibiotics)
Primary stain is malachite green flooded into a slide over a steaming water bath for 5 minutes
Rinse with water
Counterstain with safranin
Slide 12-13
What is aseptic technique?
It’s about contamination not sterility
Prevention of contamination when cultivating organisms (and containment of organisms you are working with
Requires effort as bacteria and such are everywhere
Why must the bacteria loop be heated red hot?
Cause red hot is 100s and 100s of degrees
And this temp kills all living organisms
Flames best way to keep it sterile
What are the steps to keeping a loop sterile?
Always flame loop before and after use
Too hot can kill organisms
Don’t wave loop to cool
Air currents next to flame keep that air less contamination prone
Plates should be kept inverted and opened only in use
Tubes flamed before and after use
Don’t set tube caps down
What are the 4 factors that affect growth?
Temperature
pH
Salinity
Nutrients
Each species has a range of conditions where growth is possible and more specific conditions where growth is optimal (need to mimick environment bacteria comes from)
Slide 19
What are facultative and extreme halophiles?
Facultative- can live up your nose (mucous 1% fresh but higher after drying)
Extreme halophiles- can live in 30-40% salt (dead sea)
Most bacteria need a salt concentration below 2% which is why ocean is one of least bacterially contaminating places
What are the 8 chemical needs of living organisms?
What are main 4?
Hydrogen and oxygen Carbon (backbone of most organics) Nitrogen (production of amino acids) Four main^ (HOCN) Phosphorous (for DNA RNA ATP) Sulfur (cysteine methionine) Calcium (cofactors, signalling, endospores) Trace elements (enzymatic cofactors) - don’t need to add them but worth considering (less than 0.1%)
