Course Objectives:
Cultivation of samples: growth conditions and equipment used
Identification of samples: biochemical assays & tests
... [Show More] available
Evaluation of samples: microscopy- visualization and key characteristics of specimens
Basic Equipment:
Cleaning: Autoclave 125°C- uses heat, pressure, & steam to sterilize; autoclave will take
minutes to sterilize & hot dry air which will take hours; before
opening make sure it is depressurized and will have very hot air.
Growing (bacteria/pathogens) – Fixed incubator 37°C is airtight, shelving for petri dishes
- Shaker incubator 37°C shakes culture & rotates to aerate-
liquid nutrient broth
Visualizing: microscopy
Storing: refrigerator @ 4°C to stop the growth and helps preserve samples & keeps long term
Lab Safety
1. Never eat or drink in the lab: contamination risks
2. Use PPE (personal protection equipment): gloves- latex, nitrile (purple, blue, green),thermal,
or cold gloves (liquid nitrogen): eyewear: lab coat- chemical spills
Portage Learning / BIOD 171 Microbiology Lab Notebook (2) (2) (1)-5 2021
3. Never leave lab while wearing PPE: including bathroom, hallway, or cafeteria
Outline of how to set up Notebook
Objective: To establish an organized template for keeping experimental records, procedures,
and results.
Procedure:
1. This is where each step of a given protocol is recorded
2. Be sure to clearly indicate any deviations that may occur during the experiment- put
them in red
Notes: Additional (helpful) information placed here
Results: A summary of the final outcome of the experiment should be described here
Portage Learning / BIOD 171 Microbiology Lab Notebook (2) (2) (1)-5 2021
Lab 2 Notebook
Back to Home Page
Title: KD02 Introduction to Microscopy
Objective: To learn basic parts of microscope and how to view a sample
Procedure:
1. Review the parts of microscope
2. Load sample to be viewed
3. Choose magnification
4. Adjust microscope so the sample is clear
Notes:
Parts of microscope:
1. eye pieces (ocular lens): can be pulled apart; should be able to use both eyes and see one
circle if not adjust
2. arm/neck: if moving this is what you will grab with one hand and hand under base
3. Objective lenses: this provides the magnification of the sample; shortest has the least
magnification (4x, 10x, 40x,100x)
4. Stage: the flat surface that you place your sample; holds the sample via a clamp (squeeze
together to place sample and release); stage clips raise up and lay on the glass coverslip
5. Focus knobs: located on the side of the arm/neck; Outside ring is the coarse adjustment –
makes large steps in focus; Inner ring is the fine focus- you can see the specimen but need
more detail.
Portage Learning / BIOD 171 Microbiology Lab Notebook (2) (2) (1)-5 2021
6. Iris diaphragm: located below the stage determines how much light passes through the
sample (the lever slides)
7. Base- the bottom of the microscope and should be steady and on flat surface
Load sample to be viewed:
1. Put sample on stage and make sure it is secured
2. Turn on. Left hand side you can dim or brighten the light. You can use either the knob or
the diaphragm to control the light.
Types of Objectives: dry vs oil (required on higher magnification) add a drop of oil onto slide-
helps with light refraction and lense will be embedded in the oil
3. Intensity of light source: too bright = saturation (can’t see); too dark = low visibility. Start
midway leave iris open
4. Stage guides below stage 2 knobs: top controls movement of the stage forward/backward.
Lower know moves left and right. These put specimen in range of viewing.
5. *if unsure of magnification: start on lowest power
Power of Objective X Power of Eyepiece = Total magnification: 15 mm diameter object & total
magnification is 200x larger and the diameter is 3000mm
Eye pieces are labeled with magnification on them and are removable
Coarse adjustment will raise or lower the stage- do not touch the sample with the lenses.
Look through eyepiece and slowly make adjustments. There will be a pointer inside the eye
piece you can spin the eye piece and the line will point to your sample
Results: By watching the video, I can identify the parts of the microscope and know their
basic function and how to load a sample and view it.
Portage Learning / BIOD 171 Microbiology Lab Notebook (2) (2) (1)-5 2021
Lab 3 Notebook
Back to Home Page
Title: KD03 Lab Mounting Techniques
Objective: Examination of bacterial samples through different staining techniques. Identify
samples based on color and shape
Procedure:
Dry Mount:
1. Clean slide (70% ethanol)- if it’s not clean hard to determine if you are looking at
contaminant or the pathogen
2. Circle area on slide for easy location of specimen (optional)- draw circle on bottom of slide
3. Apply organism to slide:
-If from culture, use sterile loop to apply to slide
-if from plate, use sterile loop to pick colonies and mix with a drop of distilled water
4. Air dry at room temperature until all moisture has evaporated
Wet Mount:
1. Clean slide (70% ethanol)
2. Circle area on slide for easy location of specimen using a wax hydrophobic pen – keeps water
inside ring
3. Apply organism to slide:
-If from culture, use sterile loop to apply to slide
-if from plate, use sterile loop to pick colonies and mix with a drop of distilled water
Portage Learning / BIOD 171 Microbiology Lab Notebook (2) (2) (1)-5 2021
4. View under microscope
Main reason for using wet mount is to be able to view motility of organism. Do NOT let dry out-
reapply distilled water if needed to keep organism viable. Allows to see flagella- some are not
motile and stay in a fixed location. Can be a key to diagnosing pathogen.
Gram Staining:
1. Clean slide (70% ethanol)
2. Apply organism to slide:
-use sterile loop to put 1-3 drops/slide
-spread into a thin film
3. Allow to air dry
4. Fix organism to slide by passing through flame 3 times; Do not overheat slide (takes very little
heat)
*series of dyes- depends on the bacteria it will either be absorbed or washed out
5. Rinse with crystal violet for 30 -60 seconds- dark purple dye
6. Rinse slide with water- (stains may wash out)
7. Cover with Gram’s iodine for 30-60 seconds
8. rinse with water
9. decolorize with alcohol
10. rinse with water
11. counterstain with safranin (red/pink) for 30 seconds
12. rinse with water
13. blot dry and examine
Portage Learning / BIOD 171 Microbiology Lab Notebook (2) (2) (1)-5 2021
Notes:
GRAM POSITIVE: purple
-thick peptidoglycan layer; keeps crystal violet color
GRAM NEGATIVE: pink
-thin peptidoglycan layer and is damaged by alcohol rinse & crystal violet rinsed away; pink
color is from counterstain (Safranin)
Once stained you can identify based on color/shape:
Gram positive: Purple- bacteria in chains/clusters (cocci shaped)
Gram negative: Pink- bacteria in rod shaped
Gram staining isn’t always guaranteed to identify samples
ACID FAST STAINING:
-strong resistance to decolorization
-very few structures are acid fast
-commonly used to identify mycobacterium (TB)
-carbolfuchsin dye retained (red); bacteria will remain red on blue background
NEGATIVE STAINING:
-used for organisms with opaque structures
-dark background (Nigrosin) both dye and cell membrane negatively charged so dye is repelled
- will be able to see the clusters and shapes of bacteria
WET LAB:
1. put on PPE ( gloves, eye wear, lab coat)
2. put samples in tube and ran the top of tubes through flame to sterilize
Portage Learning / BIOD 171 Microbiology Lab Notebook (2) (2) (1)-5 2021
3. cleaned slides with chem wipe
4. draw circle on slide with wax pen to keep bacteria in circle
5. flame top of tube again
6. put loop in tube and swirl to collect sample
7. spread thin layer inside circle on slide (wax keeps liquid inside)
8. let air dry
9. heat fix by running slide over flame 3 times – should be no liquid left
10. repeat with remaining 4 samples
*sterile loop should be disposed of in biohazard waste bin as well as anything else and put in
autoclave to be sterilized before disposal.
APPLYING STAIN TO EACH SAMPLE:
Supplies needed: 5 specimens, tray to dye slides in, distilled water, chem wipes, clips, timer,
crystal violet, alcohol, iodine, safranin
1. Cover entire circle with crystal violet dye for 1 min (all slides done simultaneously)
2. rinse off with water until no more color comes off
3. cover slides with iodine- apply with eye dropper don’t touch specimen (sit for 1 min)
4. rinse thoroughly with water
5. decolorization with alcohol can see some losing dye while others retain it
6. rinse thoroughly with water
7. counterstain with safranin thoroughly let sit for 45 seconds
8. final rinse –thoroughly
9. blot dry with wipe and then let air dry
*make sure to throw wipes and everything in biohazard waste bin
10. gently use wipe to make sure slide is dry –remark if wax pen came off
Portage Learning / BIOD 171 Microbiology Lab Notebook (2) (2) (1)-5 2021
EXAMINE SLIDES UNDER MICROSCOPE:
-40x magnification; stage lowered all the way
-open clips and place slide on stage to secure
-illumination set at 50%
-diaphragm open all the way
-no longer any liquid- take off eye wear
-Focus microscope:
1. raise sample to come into focus using coarse adjustment; make sure sample is in light field
2. dark image in focus use fine focus to make image clear; move stage if needed
Results:
Organism 1: (Gram +); Purple color; round cluster- Staphylococcu [Show Less]