Core Dump: Microbiol Lab Notes

Just posting some of my notes in case anyone from UNLV or CSN (or anywhere) wants  something to reference to.We had our first lab practicum mid-this-week. Some items aren't complete - differential mediums - some basic stains are explained well though.

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Practical Part:

1: Make GS and AF stains.

GS algorithm

• Saline+transferred bacteria ---> prepare smear, let air dry, then heat fix ~15s
• Add Crystal Violet, 1min, rinse with water
• Add Gram's iodine, 1min, rinse with water
• Add Gram's Alcohol, slant it**, it'll decolorize, then immediately rinse with water
• Counter stain safainin,1min, rinse with water
• Let dry with bibulous paper

AF Algorithm

• Saline+xfer bacteria ---> prepare smear, let air dry, then heat fix~15s
• Add carbolfuchsin, let stand 5min
• Add Acid-fast Alcohol, wash off last thing, then immediately rinse with water
• Add methylene blue, 1 min rinse with water
• let dry with bibulous paper
  
  

Remarks on interpretation: know result, and shape/arrangement, for an unk org.

• if Gram stain +, we should see blue-purple stains on our organisms
• if Gram stain -, we should see red-pink stains on our organisms
• G variable is??? both, dependent on age, culture mix, etc.
• if acid-fast +, reddish-purple
• If acid-fast -, (methylene blue)
  
  

//ahh, streptoooooo, ooo as in “oo” not grapes. Don't see any grape bacillus.

Cocci (little circle dots)

Coccus: o, single

diplococci: oo, paired together

streptococci: oooooooo, chains

tetrad: o^2, 4 square formation

sarcina: o^3

staphylococci: grapes*

coccobacillus: meso between cocci and bacilli

Bacilli (rod shaped bacterium)

Bacillus

diplobacilli

streptobacilli

palisades: zig-zag /|/\/\/\|/\/\/

Spirillum (spiral)

Some remarks: Basically the arrangements tend to occur when a daughter cell sticks with its parent after division. Concerning the cocci, everything is the same. Just, if the plane become irregular it becomes a “staphylo-” Nothing too special. Also note that pleomorphic may exist. Report what you see and emphasize on the most complex arrangement, many will probably exist.

Visuals:

Gram positive staphylococcus

Gram negative cocci

streptococci

Gram positive bacillus

diplobaciili

palisades

Gram negative bacillus

2: identify endospore, flagellar, and capsule stains. Structure/function

Endospore Stain:
• Impossible to see. In a strepto arrangement they were the little dots between dead cells. Stain is forced within cell, and declorization removes stain in cells except spores. A counter stain will stain the cell but not spore. So spore can be seen as a different color
• an endospore is a dormant form of a bacterium that allows it to survive poor environment conditions. Some papers also attribute it to as a form of primitive egg laying since multiple copies can be saved. Regardless, RNA is conserved. In slide you see vegetative cells and spore mother cells.

Capsule Stain

• The capsule was not stained in our slides. The stained cells were covered by nothingness, blank, nada.
• remarks: capsules are composed to mucoid polysaccharides/polypeptides that repel most stains. So they stain around the cell by the technique. Acidic stain (negative***) stains background. And the basic stain colourizes the bacteria. Thus, the capsule remains unstained and we see a white halo (the nothingness)
• Also, the function is that a capsule increases virulence in microbes. They make them less vulnerable to phagocytosis. Helps in harsh environments, can live longer on doorknobs and stuff

Endospore Stain

• stain is forced within cell, and declorization removes stain in cells except spores. A counter stain will stain the cell but not spore. So spore can be seen as a different color
• an endospore is a dormant form of a bacterium that allows it to survive poor environment conditions. Some papers also attribute it to as a form of primitive egg laying since multiple copies can be saved. Regardless, RNA is conserved. In slide you see vegetative cells and spore mother cells.
  

Flagella Stain

inject Ryu stain with needle underneath cover.

Flagella functions.. creates motility in cells.

1 tail = polar, monotrichous arrangement, amphitrichous (both ends), lophotrichous (one end ,many) peritrichous (all over, like ex)

3. differential, enriched, selective media. G+/-, for HEA, EMB, MacConkey, BAPcoliform bacteria produces gas from lactose fermentation
   selective: encourages specific, discourages other growth
   differential: allows to distinguish between diff microbes
   defined: chemicals are known and so is their composition

• undefined: composition is unknown; however ingredients are known

Below is a list of common agars: exact details are in text

• NA is w/eb
• Phenylethyl Alcohol Agar (PEA) is an undefined, selective medium that encourages G+ and inhibits growth of most G- orgs. Not considered a differential medium since it doesn't distinguish between different orgs (I question this) it's considered selective**
  
• columbia CNA /w 5% SBA: undefined, differential, selective medium. Allows growth of G+and stops/inhibits G-.
  straphylococci, streptococci, enterococci love it
• concerning blood agar. Alpha hemolytic → (eats away, leaves a green partial clearing) gamma is nonhemolytic, beta is a complete hemolytic (all RBCs are hemolyzied, but not all)
• HEA Hektoen Enteric Agar
  • selective differetial
  • selective: choose between G+, G- (allows G- grow,not so much G+)
  • differential based on G- (what species. Black color → salmonila)
• Eosin Methylene Blue (EMB)
  • complex(chem undef), slective, differential medium. Has peptone, lactose, sucrose, and dyes eosin Y /w methlyene blue.
  • Dyes inhibit G+ orgs, rxn with lactose fermenter in acidc envoinrments.
    
    

result	interpretation	“presumptive ID”
No growth (P)	inhibited	G+
Good growth (G)	Not inhibited	G-
Pink and mucoid (Pi)	Lactose ferment, lil acid	Maybe coliform
(purple, black wo green) (D)	Ferment lactose/sucrose lots of acid	X2Maybe coliform
Colorless (C)	No rxn	Not coliform

• MacConkey Agar
  • selective+differentiam medium /w lactose, bile salts, neutral red, and CY.
  • Inhibits growth of G+ orgs. Neutral red dye is a pH indicator. Red < 6.8 colorless otherwise
  • Used to isolate and differentiate members of the Enterobacteriaceae
  • remarks: if org. is a lacctose fermenter, it turns red/pink
    
    
  • 
    
    

over all remark: if it grows on Macconkey, EMB, or HEA it's G-.

Written Portion1.Magnification and Resolution. There is also contrast. We stain for this some. Impossible to get 10Kx with a light microscope (text) blue light?

2.

3.

4.

5. procedure/purpose of smear prep.

• Algorithm: emulsion
  • set-up includes the staining tray
  • place drop of water /saline on clean slide if not staining from a broth culture
  • aseptically add bacteria to water/saline. Mix and spread out.
    • Adding it to saline causes a dilute solution. smear concentration/cell densities affects stain
  • Flame it to heat fix.
    • heat fixing kills the bacteria, makes adhere to slide, and coagulates their protein for a better staining. This could distort cells, and create aerosols.
  • Let air dry, if done correctly should be cloudy
    

6. Describe G and AF stains. Justify, principles, explain in detail, step-by-step
Negative stains not discussed, but they have neg ch­arged chromogen, repelled by bacteria, and … heat fixing need not be invloved.

• gram stain is a differential stain because it divides bacteria into two classes, gram + (purple) and gram – (pink)

• cells from fresh culture are xfer to a clean slide and allowed to dry. If from agar plate, first xfer to a liquid medium for dilution. create thin, barely visible film. You must smear. fresh culture should be used since as cells age, they'll lose their ability to retain the stain. Fix cells on slide by heating (Bunsen burner, slide should feel warm afterwards)
• Stain with basic dye – crystal violet for 30-40s, then rinse to remove excess stain. Cells appear purple under microscope
• Add Gram's Iodine solution and retain on slide for ~1min. Iodine combines with crystal violet to form a dye-iodine complex. This decreases its solubility within the cell. Cell still appears purple. This means it prevents it from being dissolved?
• Decolorated by washing with ethanol or acetone. This is the differential step. G+ bacterial will retain the crystal violet, but gram negative bacteria do not. Add ethanol/acetone drop-wise, /w slide tilted at an angle. Do this until drops coming off edge start to become colorless
• WARNING: even gram + cells can lose the crystal violet-iodine complex if decloration is excessive. wash excessive ethanol with water. At this point G+ are purple and G- are colorless.
• The rinse cells are covered with a counter-stain called “safrainin” for 20-30s. This will make G- bacteria pink. Rinse with water and dry with filter paper

Theory behind G stains correlates with cell wall structure among bacterial. Ethanol is thought to shrink the thick peptidoglycan in G+ bacteria, forcing it to retain the dye. So, this thick, dehydrated peptidoglycan layer of G+ bacteria is now like “permeability barrier” this prevents the loss of the crystal violet-iodine complex.

In contrast. Since the peptidoglycan in G- bacteria is very thin and has large pores, ethanol may extract lipids and increase the cell's porosity. This will remove the crystal violet-iodine complex

again, G- cells have a higher lipid content -because of the outer layer - and thinner peptidoglycan layer than G+ cells . Also G+ cells with their thicker peptidoglycan layer have a greater degree of cross links due to teichoic acids .

Note: Iodine is not a mordant agent, it's more proper to consider it a trapping agent? Why does text continue to use “mordant”?

Acid-Fast Stain

Mycolic acid is a waxy substance that gives acid-fast cells a higher affinity for the primary stain and resistance to decolorization by an acid alcohol solution.

• two methods. Ziehl-Neelsen (ZN) and Kinyoun (K) methods. K is coldstain*. We do coldstain
• carbolfuchsin is a phenolic compound

basic stains have a negatively charged chromogen, which forms an iconic bond with a negatively charged bacterial cell.

Differential and structural stains, allow to see compartmentalizations and stuff
the acid-alcohol is (95% ethanol + 3% HCL), the carbolfuchsin would differ based on method

For Kinyoung Method

• prep smear. they recommend sheep serum vs water, but I always use saline solution. But continue on with heat-fix.
• Apply Kinyoung carbolfuchsin stain for 5min. Don't remember if it smells, but don't breath this in. rinse with water
• declorize with acid-alcohol, not to excess until run off is clear
• counterstain with something, (methylene blue) for a min. then rinse with water
• blot dry with bibulous paper or paper towels

more remarks. Do not rub with the bibulous paper - this is bad.