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"