Microlab Exam 1

Personal Protection Requirements

Wear closed-toe shoes that cover the top of the foot

Wear gloves when handling microorganisms or handling chemicals

• Do not wear gloves outside the laboratory

Wear lab coat while in the laboratory

• Do not wear lab coats outside the laboratory

Standard Laboratory Practices

Wash your hand with soap and dry with paper towels when entering AND leaving the lab. Alcohol based hand sanitizer is also available, but it is not a replacement for hand washing

Never place books, backpacks, purses, etc. on bench tops. Place them in the spaces assigned by your GTA

Electronic devices CAN NOT be kept on the lab bench after the quiz is finished. This includes, but is not limited to cell phones, tablets, laptops, and calculators

Report all cuts, scrapes, or burns to the GTA upon entering the lab. Bandages and antibiotic ointment are available in the first aid kit.

Tie back long hair

Do not wear dangling jewelry

Disinfect bench before and after the laboratory session with a disinfectant (Lysol)

Emergency procedures

The first aid kit is located next to the right front sink (facing front of lab)

The eyewash station is located at both front sinks and the sink in the back of the lab

The shower is located at the left front of the lab near the instructors desk

The fire extinguishers are located at each exit. Notify your GTA is there is a fire. Do not use the fire extinguisher except in an emergency situation

Severe Weather: The lab space is in a perfect location for severe weather shelter. It is in the basement of the building with no windows. if there is severe weather, we will shelter in place

Biosafety levels (BSL)

The Centers for Disease Control and Prevention sets guidelines for safely handling microorganisms. These guidelines consider many characteristics of the microorganisms that fall within the four biosafety levels

BSL 1 Diseases

-Microorganisms not known to cause disease in healthy adults. Examples:

• Bacillus subtilis, Serratia marcescens, Staphlococcus saprophticus

BSL 1 procedure

Standard microbiological practices are followed (SMPs) are the basic “hygiene” practices that apply to all labs that manipulate microorganisms. SMPs serve to minimize the spread of contamination generated through lab processes and to protect both personnel and the environment

Personal protective equipment (PPE) is not required

A sink must be available for hand washing

BSL 2 diseases

Indigenous microorganisms that can lead to diseases of varying severity in healthy adults

Examples: Staphylococcus aureus, Streptococcus pyogenes, Slamonella

BSL 2 procedure

Standard microbiological practices are followed

A sink for handwashing and an eyewash must be available

Laboratory access is restricted

PPE must be worn including lab coats and gloves. Eye protection and face shields are worn if needed

Work that may generate aerosols or splashes are performed in a biological safety cabinet/hood

Contaminated waste is autoclaved

BSL 3 diseases

Indigenous or exotic microorganisms that cause serious or potentially lethal disease through respiratory transmission.

Examples: Mycobacterium tuberculosis, Bacillus athracis, Francisella tularensis

BSL 3 procedure

Standard microbiological practices are followed

A hands-free sink and eyewash must be available

Locking, double door access separates the lab’s work area from other parts of the facility

PPE must be worn including lab coats, gloves, eye protection, and respirators

All work with microorganisms must be performed in a biosafety cabinet/hood

Decontamination of all waste and lab clothing before laundering

Laboratory must have negative airflow (air can not escape to surrounding rooms) and exhaust air cannot be recirculated (must be vented to outside of building after decontamination by ultra-filtration)

Medical serveillance of laboratory employees. Immunizations may be required

BSL 4 diseases

Microorganisms that are dangerous and exotic with high risk of aerosol transmitted infections. Rarely are there treatments or vaccines for these microorganisms, and the diseases they cause are frequently fatal.
Examples: Ebola virus , Marburg virus

BSL 4 procedure

Standard microbiological practices are followd

Change clothing before entering lab

A hands-free sink and eyewash must be available

The laboratory must be in a separate building or in an isolated and restricted zone of the building

All work with microorganisms must be performed in a Class lll biosafety cabinet or by wearing a full body, positive pressure suit.

Decontaminate all materials before exiting the lab (including clothes)

Shower upon exiting lab

Laboratory must have negative airflow (air can not escape to surrounding rooms) and exhaust air cannot be recirculated (must be vented to outside of building after decontamination by ultra-filtration)

Medical surveillance of laboratory employees. Immunizations may be required

Microorganism/microbe

Is a general term for living organisms that cannot be seen without a microscope, even though some people include viruses and prions in the group

Ubiquity of Microorganisms

Refers to the concept that microorganisms are everywhere. It’s not entirely true, but microorganisms (especially prokaryotes) do occupy a range of environments

In the air we breath, food we eat, and water we drink

For example, microorganisms are usually not found in our blood or cerebrospinal fluid. We invest a lot of energy in immune defenses that keep these fluids sterile (free of microbial contamination) and we get very sick when our immune defenses fail

Contamination

Is the undesired introduction of impurities like microbes, into or onto an item. This is a very real concern, since microorganisms are ubiquitous in the environment.

Agar

For microorganims that thrive on a solid surface, agar with various nutrients is poured into a petri dish. Agar is a seaweed extract used in Asian dish that melts at 85 degrees celcius and solidifies at room temp 25 degrees celcius

Angelina Heese

Petri Dish

Julius Petri made modifications of Kochs’ glass plate design in 1887. Because of Petris improvements, Koch’s glass dish became known as the Petri Dish

Contamination - laboratory viewpoint

When you grow a pure culture of a microorganism (only one type of microbe) on an agar plate or in liquid broth culture (think test tube), it is crucial that you do not introduce contaminating microorganisms to that pure culture thtough accident or sloppy lab work

Aseptic Technique

Aseptic technique is a set of precautions microbiologists take when working in the lab to avoid contamination. When you fail to use aseptic technique, you risk doing the following three things

1. Contaminate others with microorganisms that could lead to infection

2. Contaminate yourself with microorganisms that could lead to infection

3. Contaminate media and/or the microorganisms being studied in the lab

Disinfectants

You will use Lysol in this course

Disinfectants can destroy vegetative cells and viruses but may not destroy endospores. Due to this limited ability, disinfectants can not be used to sterilize surfaces or equipment.

Sterilants destroy all forms or microorganisms

Loops and Needles

Sterilized - Place needle or loop in bunsen burner flame until it is red hot. The entire wire portion of the loop or needle must be heated this way

“Flaming your loop/needle”

It is necessary to allow the loop or needle to cool before transferring an inoculum of the bacteria to be studied to ensure that your bacteria are not killed by the heat. 15-20 seconds

DO NOT touch the loop or needle to anything you included even to test if it is cool because it will contaminate it

DO NOT blow air or wave your loop or needle around to cool it

Never place it on the bench top

Inoculation

Remove cap with pinky finger - dominant hand

Briefly pass the tube through the flame

If it is liquid place loop in and twist the loop several times to deposit the bacteria

If it is a slant tube use a zig-zag pattern until reaching the top of the slant

Before closing the tube flame the mouth of the tube

Petri Plate Inoculation

Inoculating Loops are used to streak or inoculate Petri Plates

Place lid down on the bench - label the petri plate as directed

Then pass the loop into the flame until it is red hot

Retrieve your bacteria - leave the lid on the bench - turn agar towards you and gently streak the inoculum over the surface of the plate

Place the plate back onto the lid

Microscopes

The first microscope was invented in Middleburg, Holland 1590

Galileo, Hooke, Leeuwenhoek

Bright field microscopes

Simplest form of microscopy where light is either passed through, or reflected off, a specimen, Illumination is not altered by devices that change the properties of light (such as polarizers or filters). required the use of stains to visualize cells

Phase Contrast Microscopes

Converts the differences in optical density (refractive index) of cells into different shades of brightness (contrast). Allows for the visualization of morphology, external structures, and some internal structures. Stains are not required.

Dark field Microscopes

Contrast is created by a bright specimen on a dark background. It is ideal for revealing morphology and external structures , but not internal structures. Stains are not required

Fluorescent Microscopes

Use high intensity illumination to excite fluorescent molecules (fluorophores) in the sample. When a molecule absorbs photons, electrons are excited to a higher energy level. As electrons ‘relax’ back to the ground-state, vibrational energy is lost and, as a result, the emission spectrum is shifted to longer wavelengths. Fluroescence (colored light) emanates from the sample (and not the illuminating light). Required use of fluorophores

Bright Field Microscope

Is the type of microscope with which most people are familiar. They are the most popular laboratory microscope.

Light source/stage

Halogen or LED light source - found at the base as a wheel

Stage - horizontal area that supports the microscope slide is called the stage. The stage has a slide holder and stage adjustment knobs that allow for the holding and moving of the slide around the stage

Lens System: & diaphragm/condensor

Three lens: Oculars, Objectives, condenser

Three or more objectives are typically attached to a rotating nosepiece

Magnifications: 10X (low power), 40X (high dry), 100X (oil immersion).

Total Magnification = ocular power x objective lens

Condenser collects and directs the light from the light source to the slide and is located under the stage.

The amount of light that reaches the slide is controlled by the diaphragm located inside the condenser

Focusing Knobs

Bigger knob is the coarse focus knob - first brought into focus with 10X objective. After moving 40X and 100X

Smaller knob is the fine focus knob - only used for focusing

Other important terms

Immersion oil

Parfocal

Numerical Aperture

Resolving Power

Working Distance

Immersion oil - with the same refractive index as glass - used with the 100X objective, immersion oil fills the gap between the objective slide to form a continuous lens path which increases image resolution by reducing light refraction

ONLY USED WITH 100X

Parfocal - Remain in focus when changing from low power to high power objective

Numerical Aperture - A mathmatical expression that describes how the condenser len concentrates and focuses the light rays from the light source

Resolving Power - The ability of a lens system to show two closely spaced objects as distinct and separate

Working Distance - The distance between the bottom of the objective lens and the slide

Why Stain Bacteria?

Bacteria are transparent when viewed by a brightfield microscope without stain because they are composed of approximately 80% water

Single stain (simple stain) or multiple stains (Gram Stain) allows scientists to visualize cell morphology, cell arrangnement, and internal storage of materials

Smear Preparation

Every staining procedure begins with some type of smear preparation. Always come from a solid media, such as a plate or slant. Sterile water +bacteria = smears

Most Common MISTAKE - placing too many bacteria on the slide

Gram Staining

Hans Christian Gram - Danish physician - used to differentiate to major classes of bacteria

Not his intent - attempting to differ eukaryotic nuclei of long tissue

Some retained the stain (crystal violet) while others did not

More about gram stain

The deciding factor is whether a bacterium will retain crystal violet is due to the amount of petidoglycan present in the cell wall

To view Gram negative bacteria a counter stain (Safranin) is added to give them a pink/red appearance

This difference in stain uptake due to cell wall composition makes gram stain a differential stain

Must be 18-24 hours old to obtain correct results

Gram Positive Cocci

Purple stained bacteria with a spherical morphology. Gram positive cocci may occur in pairs (ex. enterococcus), in tetrads (groups of four) (ex. Micrococcus), in chains (ex. Streptococcus), or in clusters (ex. Staphlococcus)

Gram Negative Cocci

Pink to red stained bacteria with a spherical to bean shaped morphology, Gram Negative cocci may occur, in pairs (ex. Moraxella) or in tetrads (gorups of four) (ex Moraxella)

Gram positive (bacilli)

Purple stained bacteria with a rod-shaped morphology. Rods may occur singly (ex. Bacillus subtilis) or in chains (ex. Bacillus cereus)

Gram negative straight rods (bacilli)

Pink to red stained bacteria with a rod-shaped morphology. Rods occur singly (ex. Pseudomonas)

Gram Negative Curved/Sprial rods (bacilli)

Pink to red stained bacteria with a rod-shaped morphology but are also curved or spiral. Curved rods occur singly (ex. Vibrio). Spiral rods occus singly (ex. Campylobacter)

BHI with 6.5% NaCl

Differentiates microorganisms based on their ability to grow in 6.5% NaCl

Positive - Growth (turbidity)

Negative - No growth (clear)

Bile Esculin Hydrolysis

Differentiates microorganisms based on their ability to hydrolyze esculin to esculetin and dextrose. The esculetin reacts with ferric citrate in the medium to form a dark brown-black complex

Positive - Blackening of the tube

Negative - No color change or less than ½ of the slant is black

Simple and Differential

Catalase

Differentiates microorganisms based on their ability to produce the catalase enzyme. Catalase breaks hydrogen peroxide down to water and oxygen.

Positive - Bubbles

Negative - No bubbles

Coagulase

Detection of coagulase that is excreted by Staphylococcus aureus that causes fibrin clot formation

Positive - Coagulation of the plasma

Negative - No coagulation/clot formation

A excellent definitive test for confirming the identification for Staphlococcous aureus because 97% of the strains are proven to be coagulase positive - innoculating blood plasma

Hemolysis on SBA (Sheep Blood Agar)

Differentiates microorganisms based on their hemolytic activity on sheep blood agar with 5% sheep blood cells

Gamma - No discoloration

Alpha - greening

Beta - clearing

KOH test

Confirms gram stain based on whether 3% KOH can lyse bacterial cell wall and release DNA.

Gram Positive - Strings absent

Gram negative - Strings present

MSA (Mannitol Salt Agar)

Selects for microorganisms based on their ability to grow with 7.5% NaCl present in the agar and then differentiates organisms based on their ability to ferment mannitol

Positive - Growth + / Acid + Yellow colonies

Negative - Growth ± / Acid - Colorless colonies or no growth

Nitrate Reduction

Differentiates microorganisms based on their ability to reduce nitrate to nitrite or nitrogenous gas

Incomplete positive - Red coloration after the addition Nitrate A and B

Complete - No color change after the addition of Nitrate A and B and Zinc

Negative - Red after the addition of zinc

Differential

Oxidase

Diffferentiates microorganisms based on the presence of the enzyme cytochrome C oxidase in bacteria using oxidase reagent containing NNN tetra p

Positive - Any blue or purple color change within 30 sec

Negative - No color change within 30 sec

Phenol Red Carbohydrate Tubes

Differentiates organisms based on carbohydrate fermentation. This media contains specific carbohydrates and the pH indicator phenol red. When an organism is able to ferment the carbohydrate present, an acid or an acid and gas are produced and the indicator changes from red to yellow. If the carbohydrate is not fermented the color will remain red.

Positive - Red to yellow

Negative - Red

Starch Hydrolysis

Differentiates microorganisms based on their ability to hydroylze starch.

Positive - Dark agar with tan/brown clearing around streak line

Negative - Dark agar up to the streak line

Strict Aerobes

Require oxygen to grow and cannot survive in anaerobic conditions.

Facualitve anaerobes

Organisms that can grow in both the presence and absence of oxygen, but prefer oxygen when available.

Aerotolerant Anaerobes

Organisms that do not require oxygen for growth but can tolerate its presence without being harmed.

Strict obligate anaerobes

Organisms that cannot survive in the presence of oxygen and are killed by it.

Catalase - in terms of aerobes

Used by strict aerobes and facualtive anaerobes to convert hydrogen peroxide into water and oxygen.

Non-catalase peroxidases

Enzymes used by some aerotolerant anaerobes to break down hydrogen peroxide, providing protection from oxidative stress without producing oxygen.

Superoxide Dismutase

An enzyme that catalyzes the conversion of superoxide radicals into hydrogen peroxide and molecular oxygen, providing protection against oxidative damage.

Selective Medium

Allow only certain groups of microorganisms to grow and will inhibit the growth of others

Example: The incorporation of sodium chloride into mannitol salt agar (MSA) selects for the genus Staphylococcus but inhibits the growth of bacteria that cannot tolerate salt concentration

MSA and Bile Esculin are both selective and differential

Differential Medium

Contains substances that cause some bacteria to take on an appearance that distinguishes them from other bacteria

Example: When Staphylococcus aureus grows on mannitol salt agar, it ferment mannitol producing acid and changing a pH indicator in the media from red to yellow around the colonies

Ex: Sheep Blood Agar, Phenol Red carbohydrate tubes, starch agar plate

Beta hemolysis

Complete lysis of red blood cells around a colony

partial lysis = alpha

no change = gamma

Oxidative and fermentation reactions

If an organism can break down carbohydrates by oxidation or fermentation, acid is usually produced

red at alkaine pH values to yellow acidic pH values

Amylose and amylopectin

Two parts of the starch marco-molecule

(1) a straight chain of polymers of 200 to 300 glucose molecules

(2) a larger branched polymer of glucose

Starch Hydrolysis detection

Can be detected by adding Gramms iodine to a starch agar plate, a differential medium

See if you can produce amaylase to break down strach

Good tests for Stapphlococcus

MSA and coagulase

Hemolysis is a good test for

Streptococcus, Enterococcus, and Bacillus

Starch test is good for

Bacillus

Who invented the first glass dish which could hold gelatin and agar for microbial growth in 1881

Robert Koch

Bile Esculin tests for

Enterococcus and streptococcus