Lab Practical

• -Red=Gram-negative

  -Gram negative has a thick cell wall and an outer membrane which are both negatively charged so positively charged dyes stick. When ethanol is applied to the slide the outer membrane is washed away. This allows for the peptidoglycan of the cell wall to hold onto the sefran.
• -Purple=Gram positive

  -Gram positive bacteria have a thin cell wall composed of peptidoglycan. The ultraviolet stain will turn the bacteria purple and the ethanol will not wash away the color.
• Cocci=Round

  Basili= Rods

  Vibrio=Kidney

  Spirilla=Spiral

  Spirochete=Spiral
• Mono=One

  Diplo=Two

  Tetrad=Four

  Strepto=Chains

  Staph=Cluster
• Gram Stain Steps -Ultraviolet dye

  -Water

  -Iodine

  -Water

  -Ethanol 

  -Water

  -Safran

  -Water
• -Gram stain: If you want to determine if a bacteria is gram positive or negative. 
• -Simple stain: Determine if there are bacteria present and also want to view the morphology. 
• Liquid Culture:

  -Draw a circle on the slide

  -Flame sterilize loop and test tube

  -Take a loopful of bacteria

  -Put the loopful on the slide

  -Flame sterilize loop and test tube

  -Allow to completely dry

  -Heat fix
• -Draw a circle on the slide

  -Flame sterilize loop 

  -Put one loopful of water onto the slide

  -Flame sterilize loop 

  -Transfer a small amount of bacteria to the slide

  -Flame sterilize loop

  -Heat fix
• -Heat Fixing kills the bacteria and adheres them to the slide. 

  -Allow the slide to completely dry

  -Pass the bottom of the slide over the flame of the Bunsen Burner 3-4 times
• -A monolayer allows to kill the bacteria, heat fix, stain and visualize the morphology. 
• -The difference between making a smear from a broth and a plate is that from a plate you have to mix water with the bacteria making a broth. 
• -A pure culture has 1 type of bacteria. 
• -A mixed culture has more than 1 type of bacteria. 
• -A contaminated culture is one that has more types of bacteria than there are suppose to be. 2 types of bacteria show up on the streak plate when there is supposed to be one. 
• Plaques have a dot in the middle surrounded by a ring of no growth. Colonies are a dot of growth. 
• In CFU you are counting the colonies on a plate. In PFU you are counting the plaques that have formed. 
• The neat is the 1st, it is not diluted, it is a pure culture. For example, the e-coli before it has gone through its dilution process. 
• -A dilution is a where you make a substance less concentrated. 
• -Serial dilutions are where you mix a sample with saline, then take that combination and put it in more saline. This will cause more dilution and less concentration. Serial dilution makes it easier to count microbes and then calculate the original CFU/mL
• The red pipette is 1ml=1000ul
• -Total magnification=Objective Lens (__x)  (10x)
• -Depending on the size of the organism determines the objective lens, the lowest is scanning which is 10x and the highest is oil which is 100x. 
• -Eye piece=look into the microscope
• -Nose piece= adjust the objective lens
• -Objective lens= adjust the magnification
• -Stage clip= hold the slide in place
• -Diaphram=adjust amount of light going through the microscope
• -Arm=allows for the microscope to be carried
• -Stage= the slide sits on it, moves up and down by using the course adjustment knob
• -Course adjustment= moves the stage up and down
• -Fine adjustment=define the image of the microbe
• Stage control=allows to move the slide around so it is in view
• -Base= the microscope sits on the table with it
• -Length of field of view in um/# of cells that will fill across the field of view
• -40x=4mm

  -100x=2mm

  -400x=0.5mm

  -1000x=0.2mm
• Resoulution is the length between objects required to determine that they are two separate objects. By adding oil and by using light that has a shorter wavelength increases the resolution. 
• -Stage is completely lowered

  -Adjust to 4x objective using course knob

  -Adjust to 10x objective using course or fine adjustment knob

  -Adjust to 40x objective using a fine adjustment knob

  -Apply oil to slide

  -Adjust to 100x objective using a fine adjustment knob

  -Move 4x into place

  -Lower stage

  -Clean up the oil
• -The wet mount has a divot that allows you to observe motile organisms
• -Fixed smears do not allow for motility