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How to properly prepare a
bacterial smear (3.5)
- 1. Get a clean slide, draw a circle on the bottom of it.
- 2. Sterilize inoculation loop using bactincinerator.
- 3. Place 1 loop of water on slide and eliminate moisture on loop using heat.
- 4. Tap petriplate of micrococcus luteus gently and mix into water drop.
- 5. Spread and make a thin film, let air dry.
- 6. Sterilize innoculation loop.
- 7. Heat fix slide: use heat in 30 seconds intervals; "coffee-hot"
- Aseptically: without contaminating culture, the sterile medium or the surroundings.
- Contaminant: unwanted microbes
- Sterile: All microbes are destroyed or removed
How to Heat Fix:
How: Warm slide of over the bacticinerator in 30 second intervals, until the slide is hot to the touch (coffee-hot)
- Advantages: You get the bacteria to stick to the slide. so when you drop dye on it, the bacteria won't fall off
- Disadvantages: Distorts the cell shape
Simple Stain Procedure (3.5)
Purpose: to highlight the entire microorganism so that cellular shapes and basic structures are visible
- 1.Take slide with prepared smear, clear workspace.
- 2. Place 2-3 drops of methylene blue dye
- over smear.
- 3. Allow slide to air-dry for one minute.
- 4. Gently rinse slide in stain tray with de-ionized water from a squirt bottle.
- 5. Blot dry using bibulous paper booklet, don't rub.
- 6. Clean workspace before getting the microscopes out: use 100x lens + oil
What a basic dye (stain) is
and examples of basic dyes.
The color of these dyes are in the positive ion
ex: Methylene blue, safranin, and crystal violet
Cyanobacteria & Algae:
-what winogradsky level?
-energy source?-what winogradsky level?
-what winogradsky level?
Autotrophic Chemolitho(Auto)trophs: Inorganic chemicals, inorganic carbon source
In Aerobic soil, below water
Green and Purple Non Sulfur Bacteria
-what winogradsky level?
Photoheterotrophs: light and organic carbon source
microaerophillic soil; below aerobic soil and above anaerobic soil
Green and Purple Sulfur Bacteria
-what winogradsky level?
Photoautotrophs: light and inorganic carbon (Hydrogen sulphide provided by desulfibriobacteria below)
in Anaerobic Soil; below microaerophillic soil and above anaerobic nutrient soil
-what winogradsky level?
Sulfur Reducing: converts sulfate to Hydrogen Sulphide SO42- ===> H2S
Bacteria that uses organic compounds in Fermentation to produce organic waste.
Found in anaerobic nutrient (sulfate/carbonate soil)
Use CO2 to make methane (in Archea)
Found in nutrient anaerobic soil ( sulfate and carbonate)
Spyrogyra (Pond Org)
- Part of Algae group: contain distinctive spiral chloroplasts.
Diatoms (Pond Org)
- Photosyn unicellular Prokaryotes:belong to ALGAE group.
- Either centric or pennate (billaterally symetrical and elongated)
- Pigment: fucoxanthin--gives off a golden brown hue.
Giardia Lambia Cysts:
--> what is a cyst?!
--> where is it found, how is it transmitted?
inactive form, non-motile and resists DISINFECTION.
-oval shape, 4 nuclei
Shed in Feces, transmitted in fecally contaminated water and by oral contact of food.
--> what is a Trophozoite?
--> common where?
- Trophozoite: Active-Feeding form
- Most common in duodenum; also found in stool
- Has two nuclei with karyosomes and axostyle-a line in the middle of the cell and 8 flagella
Plasmodium Falciparium (ring stage)
- Transmitted by mosquitos;
- cause malaria: fatal vessel obstruction and thrombosis.
- --sporozoite transmitted my mosquito bite
- --reproduces in liver THEN RBC's
- Ring stage-parasite in RBC.
Most found in vaginal area, transmitted sexually- STD: trichomoniasis
Shape&Arrangement: grape-like round clusters
Acid Fast: Stains blue--methylene blue counterstain (non-acid fast)
Gram Stain: PURPLE(crystal violet=Primary stain)
Gram? (and is it affected by lysozymes)
Shape/arrangment: Round cells, tetrads
-->colored blue (basic methylene blue stain: dye has positive charge and is attracted to negative cell walls); in simple staining method
--> GRAM Positive! YES! (b/c directly exposed to lysozymes which hydrolyze covalent bonds of NAG +NAM in peptidoglycan, no outer lipid membrane to protect it.)
- Shape/arrangment: Rods, no arrangment
- --stained PINK (gram negative cells)
- --Gram Negative;
- --NOT Affected by Lysozymes: because it's peptidoglycan layer is sandwiched between outer lipid membrane and inner membrane
Shape&Arrangement: Rods, no arrangement
Acid Fast: Stained Red-Carbolfuchsin
- 1. Disinfect the table before beginning work, wash hands before and after.
- 2. Tie back long hair near heat
- 3. Testubes go upright
- 4. Keep flammable items away from heat: stains, reagents, papers
- 5. Put away stains before microscopes
- 6. Incubate petri dishes upside down, label agar side
If you spill live bacterial cultures:
- 1. Notify instructor.
- 2. Cover spill with a paper towel and pour disinfectant on it.
- 3. Let it sit for 15 mins then morgue spilled culture.
- 4. Wash hands with soap and water.
- 5. After 15 mins: wipe up the spill with clean paper towels. Use bleach then clean with more paper towels.
- 6. Discard and wash hands.
1. Petri dishes go in morgue: biohazard container
3. Contaminated sharps (such as as used Pasteur pipets and slides of live bacteria) in small red biohazard sharps container
- 2.All reusable glassware must be prepared properly and placed in morgue:
- -remove tape from test tube, loosen screws
- -tubes go upright in rack in morgue.
- -bottles/flasks go in desginated morgue area
Microscope parts & functionss:
--> Oil immerson:
**Oil immersion: l
- Ocular lens: 10 x (eyepiece)
- Nose Piece: Holds the objective lens
- Objective Lens: 4x, 10x, 40x, and 100x
- Course Focus Knob (10x)
- Fine Focus Knob (40 x)
- stage: The horizontal platform that supports the microscope slide.
- Iris Diaphragm: It regulates the amount of light that reaches the slide
- Condensor: focuses the light from the source onto the specimen
imits light refraction.
Types of Light Microscopy:
- Phase Contrast
Use stains, and light hits the specimen directly.
- Image appears light against a dark background
- Opague disk: next to condenser, blocks all light from hitting speciment dirrectly (light must go around disk and bounce off speciment)
- -Only light that is reflected by specimen is seen
- -Can be used with living/unstained cells (image is dark in background)
Phase Contrast Microscopy:
-two types of light rays?
-improve contrast by:
Provides greater contrast for internal structures of specimen, can be used with living/unstained cells
-Direct light ray: goes directly through the speciment (no bending)
- Diffracted light ray: these are bent by interaction with the specimen and "slow down" by 1/4 wavelength (eyes dont' pick up difference)
improve contrast by: annular ring and phase/diffraction plate--> uses destructive interference to make cell structures appear darker by speeding/slowing down some rays.
Used for viruses/small bacteria, small beam of electrons bounce off of the specimen, increased resolving power (shorter wavelength).
Limitations: can't look at living specimens, requires specialized training and is expensive.
Types of Electron Microscopy
1. Transmission electron Microscopy: Requires a very thin section of specimen stained with heavey metal, electron bean goes through specimen and image appears flat.
2. Scanning Electron Microscopy: Entire specimen is covered with gold (heavy metal) and electron bean scatters electrons on specimen. Get 3D images.
Colony vs. Culture
Colony: group of microbes on a plate that are all identical-just one type of species
Culture: All bacteria on a plate (could be mixed or pure)
Quadrant Streak Isolation Plate
--> Method (two days)
- Purpose: To create pure culture from a mixed culture and isolate one species for research
- by using the Quadrant Streak Isolation Plate Method.
Day 1 (02.26.13)
- 1. Label a sterile agar plate with
- numbers: 1-4 on each quadrant.
- 2. Obtain a sample of mixed culture with
- a sterile loop.
- 3. Starting at the 1st quadrant’s
- edge, lightly drag the loop side to side in a zig-zag motion (streak) until you
- cover one-third of the plate.
4. Remove and sterilize the loop for 6-7 seconds before allowing it to cool.
- 5. Rotate the plate 90 degrees and at
- the edge of the second quadrant, begin streak by intersecting first 2-3 lines
- with previous streak before extending away from it.
6. Remove and sterilize the loop again.
- 7. Rotate petri dish 90 degrees to the
- third quadrant, repeating third streak pattern to intersect first 2-3 lines
- with the second streak before extending away from it.
- 8. Finally rotate the dish 90 degrees to
- the fourth quadrant and perform a fourth streak, intersecting with the third
- quadrant streak and then extending into the center of the plate.
- 9. Sterilize the loop and incubate the
- closed plate in an inverted position for 48 hours.
Day 2 (02.28.13)
- 1. Obtain incubated Streak Isolate Plate,
- choose one isolated colony and touch it gently to get bacteria on a sterile loop.
- 2. Transfer the pure culture to a new
- streak isolation plate.
- 3. Follow the same procedure in creating
- a quadrant streak isolation plate from day one.
4. Label and Incubate plate upside down.
Biofilms (ch 1)
- complex aggregation of microbes; slimy (on your teeth)
- Often resistant to antibiotics
Three Staining Techniques:
- Differential ( Gram & Acid-Fast staining)
aqueous or alcohol solution of a single basic dye.
Purpose: to highlight the entire microorganism so that cellular shapes and basic structures are visible; applied to a fixed smear for a certain length of time, washed then dried.
>>ex: methylene blue, carbolfuschsin, crystal violet, and safranin
-Primary and counterstains used?
-Gram positive vs. Gram Negative cells
- Primary=Gram Positive: crystal violet (purple)
- Counter =Gram negative: safranin (pink)
: enhances crystal violet staining by forming a crystal violet-iodine complex (CVI)
: the acetone extract the thicker lipid content in gram negative cells to make it more porous and incapable of retianing the CVI complex
Gram Positive cells:
thicker peptidoglycan and greater degree of cross linking by tichoic acids which trap the CVI complex and doesnt get decolorized (stays purple)
Gram Stain Purpose and Procedure?
: a simple staining test that simply classifies two main groups of bacteria. Gram positive, and gram negative based on cell structure.
- 1. Take a clean slide and label your Initials,“Gram Stain”. On the back of the slide, use permanent marker to draw a large circle in the middle.
- 2. Sterilize loop and add one loop of water to the front part of the slide (inside the circle).
- 3.Sterilize loop. Tap into Staphylococcus Epidermidis sample and inoculate the water drop on the slide. Mix smear evenly.
- 4.Sterilize Loop. Tap loop into Pseudomonas Aeriginosa and mix into smear.
- 5.Allow smear to air dry and then heat-fix until slide is “coffee-beverage” hot to touch.
- 6. Begin Gram-Stain procedure by covering the smear in 2-3 drops of crystal violet. Wait 20 seconds before rinsing slide with deionized water over the stain tray.
- 7.Add 2-3 drops of Iodine to cover the smear and allow it to sit for 1 minute. Do NOT rinse it but just tilt slide and allowstain tray to catch excess iodine.
- 8. Allow Alcohol (the decolorizer) to trickle over the smear for 3-5 seconds (time-sensitive part) and immediately rinse with distilled water.
- 9.Then cover smear in 2-3 drops of safranin stain (counterstain) for 1 minute before rinsing with distilled water.
- 10.Use bibulous papers to blot dry the slide.
- 11.Clean work area of gram-stain kit before observing slide under oil immersion.
Most useful-classifies bacteria into two large groups-gram positive and gram negative.
- 1. heat-fixed smear covered with crystal violet for( Primary stain: gives its color to all cells).
- 2.Then dye is washed off, smear covered with iodiine and washed off so that both gram positive and negative bacteria appear dark violet.
- 3. Slide is washed with alcohol solution: decolorizing agent-removes purple from cells of certain species.
- 4. Alcohol is rinsed off ==> slide is stained with safranin (basic red dye). Blot dried.
Bacteria that retain the dye after decolorizing alcohol
-->thicker peptidoglycan cell wall,dye and iodine enter and form CV-I complex-larger molecule than crystal violet molecules that entered, so it cannot leave==> so alcohol does not wash out the dye.
Bacteria that LOSE the dark violet color after decolorization
must be added to stain these pink to counter the original purple stain so called a "counterstain"
- --> contain layer of lipopolysaccharide as part of cell wall (alcohol wash disrupts this outer layer, and the "CV-I" complex is washed out through the thin layer of petidoglycan)
- -->must be redyed with safranin. :)
Gram positive vs. Gram Negative walls (image)
another differential stain
-->differentiates into distinctive groups by binding strongly only to bacteria that have a waxy materia
l in their cell walls.
Myobacterium tuberculosis & M.leprae(leprosy) and both pathogenic, and Nocardia.
- 1.Carbolfuchsin (red dye) applied to fixed smear slide THEN heated (enchances penetration and retention of dye).
- 2. Let slide cool, water rinsed.
- 3.THEN slide is given acid-alcohol (decolorizer: removed red stain from bacter that are NOT acid-fast).
- **Note: carbolfuchsin is more soluble in cell wall lipids than acid-alcohol, bacteria that lack lipids in their cell wall will have the red color rapidly removed--> leaves it colorless.
THEN smear is stained with methylene blue
(as a counter stain)
so that non acid-fast cells become blue.
Acid Fast Stain Purpose and Procedure:
- Purpose: To Perform an acid-fast stain to
- identify Myobacterium.
- 1.Prepare a clean slide by drawing a large circle on the backside with a permanent marker.
- 2.Sterilize the loop in the bacticinerator and add 2 loops of Staphylococcus epidermidis (in liquid form already, no need to add water) to the front of the slide—within the circle.
- 3.Sterilize the loop again. And tap loop in the slant to get one loop of Myobacterium smegmatis (tan culture on a blue medium).
- 4.Mix loop of M. smegmatis bacteria with previous S. epidermidis drops on slide to create a smear. Spread smear evenly.
- 5.Allow to dry and heat-fix the slide over the bacticinerator until warm to touch (“coffee-beverage”)
- 6.Place slide on stain rack and place a small square paper towel over the smear. Soak paper towel with 2-3 drops of Carbolfuchsin dye. Let towel sit over smear for 20 minutes. Check every now and then to maintain moisture of the towel, adding more dye when the paper dries.
- 7.After 20 minutes, rinse with deionized water.
- 8.Add 2 drops of Acid-alcohol decolorizer and immediately rinse with water (time sensitive).
- 9.Add Methylene blue (counterstain) and allow it to sit on smear for 1-2 minutes before rinsing with water.
- 10.Use bibulous paper to blot-dry the slide.
- 11.Clean work-space and observe slide under oil immersion.
microbe communities; in slime
(matrix of polysaccharides with DNA/proteins)
- -quorum sensing: allows bacteria to coordinate activity and group together
- ex: plaque, pond rocks
- -work cooperatively on same task
- -primitive circulatory systems: incoming nutrients and waste channels
- -1000x More resistant to microbicides; form on medical devices
- -protected by antibodies, WBC phagocytosis and antibiotics
: block quorum sensing, incorporate antimicrobial into possible biofilm surfaces; must physically remove them
Lysozymes Purpose and Method:
- Purpose: To
- observe the effects of lysozymes upon peptidoglycan within the cellular walls
- of Micrococcus luteus and Serratia marcescens
b. Then add two drops of Egg white per plate.
c. Incubate the plate right side up.
- a. First prepare bacterial lawns for both Micrococcus luteus and Serratia marcescens (both are broth cultures):
- 1. Take a sterile swab and dip it into the broth culture test-tube until well-soaked.2. Take cultured swab and gently slide over a sterile agar plate. Make sure to swab evenly, covering the edges and avoiding gaps.3. Take a quarter turn of the petri plate and swab evenly over the medium to create a perpendicular spread that covers the plate.4. Take a second quarter turn and repeat and swab over the plate. 5. Repeat two more quarter turns with perpendicular swab patterns until 360 degrees has been completed.6. Throw away the used swab in the bleach container.