BIO Lab Practical
Gram Stain
- Most commonly used differential stain in microbiology. •Developed by Christian Gram in 1884 •Essential tool for classification and differentiation of microorganisms by separating G(+) vs. G(-) •Requires fresh cultures. As cultures age, cell walls deteriorate and results will not be accurate. Gram variability!
EMB pt.2
1. Non-fermenters will be light pink or translucent (blend with the agar) and the agar will be maroon 2. Organisms that produce weak acids from fermentation of lactose will have maroon colonies on maroon agar 3. Organisms that produce strong acids from fermentation of lactose will have METALLIC GREEN colonies on maroon agar
MacConkey's Agar Plate pt.2
1. Non-fermenters will be tan and the agar will be tan 2. Organisms that produce weak acids from the fermentation of lactose will have pink colonies on tan agar 3. Organisms that produce strong acids from the fermentation of lactose will have pink colonies with a pink halo in the agar
Gram-Stain Procedure
1. Primary stain (Crystal violet- a basic stain) 2. Mordant (Gram's iodine- Stain enhancer) (something that helps fix dye on or in a cell but does not itself contribute color) 3. decolorizing agent (Ethanol 95%) 4. Counterstain (Safranin- a basic stain)
Pipetting
1. disposable tip attachment point 2. tip ejector shaft 3. tip ejector button 4. volume adjustment knob 5. plunger 6. volume indicator window
Basophils
A circulating leukocyte that produces histamine.
endospore stain
A differential stain used to detect the presence and location of spores in bacterial cells. -Schaeffer-Fulton Method
Neutrophils
A type of white blood cell that engulfs microbes by phagocytosis
Lymphocytes
A type of white blood cell that make antibodies to fight off infections
Microaerophiles
Don't like oxygen but can tolerate some but also need it.
Facultative Anaerobes
Don't need oxygen but aren't harmed by it, basically the strength of the previous two but none of the weaknesses.
Blood Agar Plate
PlateMade typically from 5% Sheep's Blood(Enriched Media) Used to grow fastidious organisms(Require very specific nutrients or conditions) Alpha Hemolysis: Partial lysis of red blood cells Beta Hemolysis: Full lysis of red blood cells(clear zone in media) Gamma Hemolysis: TRICK WORD no hemolysis happening.
Bacilli (bacillus)
Rod shaped bacteria
Transformation
The process of horizontal gene transfer by which bacteria pick up genetic material from the environment
Bibulous Paper
a special absorbent paper used to dry slides; blotting paper
Eosinophils
a white blood cell containing granules that are readily stained by eosin.
How to bring the slide into focus
coarse adjustment knob
how to bring the slide into sharp focus
fine adjustment knob
Mycobacterium leprae
leprosy
Mycobacterium spp.
•Aerobic Gram positive small bacilli •Form dry, crusty growth pattern on slants •Present in soil and water. Some species are pathogens •We will be using Mycobacterium smegmatis, aBSL-2 organism found in soil and water.
streak plate technique
•Commonly used to separate different types of bacteria from the mixed culture. •Mixed culture vs. pure culture
Presumptive Test
•Determines the presence of coliforms in a water sample and determine the probable number of such organisms (the MPN-most probable number) •-Add water to lactose broths with Durham tubes. Presence of acid and gas indicates presumptive evidence for coliforms. •Suggests that water may not be suitable for drinking
Electronic cell counters
•Fast •Easy to use •Machine is expensive •Counts ALL particles of a specific size.
Staphylococcus epidermidis non-acid fast
•Gram positive cocci in clusters •Colonies are white, circular, convex with entire margins •Facultative anaerobe, moderate halophile that is resistant to drying, heat and sunlight •BSL-1-Can cause nosocomial infections. Among most common blood infections. Highly prevalent among neonates. Leading cause of Intravascular Catheter Infections and 40% of valve prosthetic endocarditis
Rules for transferring
•Inoculating needles are always used to transfer bacteria from a slant or broth to a deep •Inoculating loop are always used to make a streak plate •Either a loop or a needle can be used to transfer bacteria to a broth. Can't hurt a broth
Bacillus genera
•Large, Gram positive rods, aerobic or facultative anaerobic organisms •Bacillus anthracis •Anthrax(3 Different types) •Causes Black lesions. After death there is a lack of rigor mortis. •Bacillus cereus •Most common source of food poisoning
Eosin Methylene Blue (EMB) Agar Plate
•Maroonwhen you look at the SIDE of the plate •Sugar is lactose •Eosin Y and methylene blue inhibit the growth of SOME Gram (+) microbes •Selectivefor Gram(-) organisms •Some Gram (+) organisms will grow (weakly) •Differentialfor lactose fermentation
Serial Dilution Viable Plate Count
•Most frequently used method of measuring bacterial populations •Count only live cells •One bacterium grows to form one colony hence CFU(colony-forming unit) •Takes 24-48 hours
Direct Microscopic Count using a Petroff-Hauser cell counter
•Relatively inexpensive •Quick •Requires high # of bacteria 106/ml •Count live and dead cells
Sporulation
•Sporulation is the formation of spores often triggered by depletion of carbon and nutrient sources •It is a survival mechanism •Spores are resistant to heat, drying, freezing, UV radiation, and chemical agents •During sporulation, cell undergoes changes to encapsulate its DNA and ribosomes within a central core
Clostridioides organisms
•Strict anaerobes, found the intestines of mammals •Clostridioides difficile •Highly contagious gastrointestinal disease •Exceptionally resistant to antibiotics due to its ability to form long lasting spores
Germination
•The process in which a free spore revert to a metabolically active cell •It happens when the environment is again favorable for cell growth •Remember: Sporulation and germination are NOT methods of reproduction.
PEA agar plate
•Uses Phenylethyl Alcohol to select MOSTLY Gram Positives. Not Perfect!! •Often an addition to blood agar plates. •A is a PEA plate (selective) •B is a TSA plate (non-selective)
MacConkey's Agar Plate
•Violet when you look at the SIDE of the plate •Sugar is lactose •Bile salts and crystal violet which prohibit growth of Gram(+) microbes •Neutral Red is the pH indicator •Selective for Gram(-) organisms •Differential for lactose fermentation
Microorganisms in water
•Water can be contaminated with bacteria, viruses, or protozoa. •Escherichia coliis used as a sentinel organism, one that alerts officials to the presence of potentially pathogenic organisms
Cocci
sphere shaped bacteria
Spirillium/spirilla
spiral shaped bacteria
subculturing
the process of transferring organisms from one medium to another following all aseptic techniques
quality control
the strategy for minimizing errors by managing each stage of production
Oxygen Requirements
- zone 1: RequirementsAtmospheric Oxygen (~20%) - zone 2: Limited Oxygen (2-10%) - zone3: Oxygen levels less than 2%
Mycobacterium tuberculosis
tuberculosis
Simple stains
use only one dye to stain bacteria. Provide information about cell morphology and arrangement.
Differential stains
use two or more dyes. Provide more details about cell wall properties such as Gram (+)/(-) and presence of mycolic acid.
Negative Stains
uses nigrosine dye to stain the slide background leaving bacteria clear due to similar charges on the dye and bacteria. Used for bacteria that can't be heat fixed and electron microscopy. Bacilli, Spirillum, Cocci
Acid Fast Stain
Acid Fast bacteria have a waxy coating of mycolic acid. This resists most stains unless heat is applied. Heat will force the dye through the mycolic acid and remain on the cell even under acid conditions, thus acid-fast. -ZIEHL-NEELSEN METHOD
Monocytes
An agranular leukocyte is able to migrate into tissues and transform into a macrophage.
Mannitol Salt Agar Plate
Bright Red when you look at the SIDE of the plate •pH indicator is phenol red •Sugar is mannitol •Contains 7.5% salt •Selective for Gram (+) Halophiles •Differential for mannitol fermentation •fermenters turn the agar yellow •gram-positive halophiles that do not ferment mannitol will grow, but the agar stays pink. Colony color will vary.
Aerotolerant
Can handle some oxygen but doesn't use it to live
Estimating Bacterial numbers by use of a Spectrophotometer
Can make a graph of absorbance vs. time and match that with plate counts set up at the same time with the same culture Indirect estimate Requires 10-100 million bacteria to register turbidity
Heatshock
Extracellular DNA enters the cell - it started at 4 degrees Celsius - 42 degrees celsius water bath - returns back to 4 degrees celsius - broth 37 degrees celsius
Lab Safety: PPE
Goggles, Nitrile Gloves, Full Length Lab Coat
EC-MUG
MUGSelective media for fecal coliforms. MUG(4-methylumbelliferyl-ß-D-glucuronide) is cleaved by glucuronidase enzyme produced by E. coli.
Viable Plate Counts
Methods used to determine the number of live bacteria in a sample
Gram Stain Bacterial Slides
Micrococcus luteus Bacillus subtilis Serratia marcescens
Aerobes
Need Oxygen or else dead
human blood smear
Neutrophil, monocyte, Basophil, Lymphocyte, and Eosinophil
Strict Anaerobes
No Oxygen or else dead
Setting volume levels
P20, P200, P1000
Colony Characteristics
Size, Pigment, Form, Margin, Elevation - View chart - Serratia marcescens and Escherichia coli
results of sporulation
Sporangium = vegetative cell (pink) Endospore is retained within the sporangium (green) Endospore locations: -Central -Terminal -Subterminal (most of what we are going to see) -Free spore ( older the culture will have free spores)
Spore forming organisms
Spore forming organisms are Gram positive rods Bacillus genera Clostridium genera Clostridioides genera
Clostridial organisms
Strict anaerobes, found in soil, dust Clostridium botulinum Type of food poisoning =botulism produces neurotoxin responsible for flaccid paralysis - used for botox Clostridium tetani - "lock jaw"