MICROBIOLOGY-1.txt

Work area is treated with a disinfectant that will kill any microorganisms that may be present, but not endospores

A loop or needle is sterilized by inserting it into a Bunsen burner flame until it is red hot, ensuring that all contaminating organisms will be incinerated. Allow the loop/needle to cool completely before picking up any inoculum, this will ensure that all viable cells are transferred.

The cap is removed, and the mouth of the tube is passed through the flame. If the tube is a broth tube, the loop is inserted into the tube and twisted several times to ensure the organisms on the loop are deliverd to the liquid. If the tube is an agar slant, the surface of the slant is inoculated by drawing the loop up the surface of the slant from the bottom of the slant to the top. For stab cultures, a needle is inserted into the agar medium. After the culture is inoculated the mouth if the tube is reflamed, and the tube is recapped.

After completing the innoculation the needle/loop is flamed in the bunsen burner, to destroy any organsims. Iteams should be returned to their appropriate place, never laid on the desk.

Loops are used to inocutlate or streak petri plates. The plate cover is raised andheld diagonally over the plate to protect the surface from any contaminationin the air. The loop containing the inoculom is then streaked gently over the surface of the agar

Liquid(broth), Semi-solid(slant), Solid (plate)

My Initials, Table Number, Instructors Name, Name of Organism, Media

48 Hours of Incubation at 37 degrees Celsius

0= no growth, + is 1 to 10 colonies, ++ is 11 to 50 colonies, ++++ is over 100 colonies. Bacteria growth appears as small dots, each dot representing a colony. If the dot posses any fur, or fuzziness, it is mold.

With a swab, moisten it in distilled waterand rub the swab over a part of your body. To expose the agar plate, couch into it, or rub the swab over the entire surface of the plate.

• REMOVING ORGANISMS
• 1.) Inoculating loop is heated untill red hot.
• 2.) Organisms in broth culture are dispersed by shaking tube.
• 3.) tube enclosure is removed and mouth of tube is flamed.
• 4.) A loopful of organisms is removed from tube.
• 5.) Loop is removed from culture and tube mouth is flamed
• 6.) Tube is enclosure is returned to tube.
• INOCULATING NUTRIENT BROTH
• 7.) Cap is removed from steril broth tube mouth is flamed.
• 8.) Unheated loop is inserted into tube of steril broth.
• 9.) Loop is removed from broth and tube mouth is flamed
• 10.) Tube enclosure is returned to tube.
• 11.) Loop is flamed and retuurned to receptical.

• 1.) Inoculating loop is heated untill it is red hot.
• 2.) Cap is removed from slant culture and tube mouth is heated
• 3.) Organism is picked up from slant with inoculatin loop.
• 4.) Mouth of tube is flamed. Inoculating loop is not flamed.
• 5.) Slant culture is recapped and returned to test-tube rack.
• 6.) Tube of steril agar slant is uncapped and mouth is flamed
• 7,) Slant surface is streaked with unflamed loop in serpentine manner
• 8.) tube mouth is flamed, recapped and incubated.
• 9.) Loop is flamed red hot and replaced in reciptical

• 1.) Inocluating loop is heated until red hot.
• 2.) With free hand, raise the lid of the petri plate just enough to access a colony to pick upa loopful of organisms.
• 3.) After flaming the mouth of a sterili slant, streak its surface, usually in a fishtail method.
• 4.) Flame the mouth of the tube and recap it.
• 5.) Flame the inoculating loop and return it to the receptical

• 1.) Inoculating loop is heated untill it is red hot.
• 2.) Cap is removed from slant culture and tube mouth is heated
• 3.) Organism is picked up from slant with inoculatin loop.
• 4.) Cap is removed from steril broth tube mouth is flamed.
• 5.) Unheated loop is inserted into tube of steril broth.
• 6.) Loop is removed from broth and tube mouth is flamed
• 7.) Tube enclosure is returned to tube.
• 8.) Loop is flamed and retuurned to receptical.

Wash the slide with soap or Bon Ami and hot water. Handle the slide by the edges

• 1.) Shake the culture tube to suspend the organism
• 2.) Heat the loop
• 3.) Flame the neck of the tube containing the organism
• 4.) After allowing the loop to cool for at least 5 secs, remove a loopful of organism.
• 5.) Flame the mouth if the tube again before replacing the cap

• 1.) Two loopfuls of water are placed on the target circle of the slide
• 2.) A very small amount of the organism is dispersed in the water over the entire target circle area using the inoculating loop.
• 3.) The smear is allowed to air dry at room temperature
• 4.) Slide is passed throug the flame several times to heat-kill and fix the organisms to slide.

• 1.) Two loopfuls of liquid containing organisms are placed in the center of the "target circle"
• 2.) Organisms are over entire area of the target circle
• 3.) The smear is allowed to dry at room temperature
• 4.) Slide is passed through flame several times to heat kill and fix organisms to slide.

The fact that bacteria are negatively charged produces a pronounced attraction between these cationic chromophores and the organism. These dyes are basic dyes

The basic dye used in this technique is Methylene blue (methylene+ chloride-) Some other common basic dyes are Basic Fuchsin, Crystal Violet. These dyes posses chromophores, which are color bearing ions that are positively charged

Those dyes that have anionic chromophores are called acidic dyes. Eosin (sodium+ eosinate+) is one. The dye will not stain bacteria because of the electrostatic repelling forces that are involved. Instead it is the background that is dyed rather than the organism

• Small, nonmotile, irregularly staining pleomorphic
• Gram-positive rods with club-shaped swelled ends but no spores; may be straight
• or slightly curved.
• Cells tend to lie parallel to one another (palisades) or at acute angles (coryneforms),
• due to their snapping type of division
• Vary greatly in dimension, from 0.3 to 1 um in diameter and 1.0 to 8.0 um in length

• 1.) A bacterial smear is stained with methylene blue for one minute.
• 2.) Stain is briefly washed off slide wih water
• 3.) Water drops are carefully blotted off slide with bibulous paper

• average size, is 1.1 to 1.5 um wide by 2.0 to 6.0 um long.
• Morphology is rod shaped
• Arrangement is peritrichous

The dye we use in this exercise is Methylene blue. We could also use Crystal violet or basic fuchsin

• Simple Stain:
• A simple stain consists of a solution of a single dye. Some of the most commonly used dyes are methylene blue, basic fuchsin, and
• crystal violet. Simple stains allow one to distinguish the shape (morphology) of the bacteria. For example, E. coli and Bacillus Subtillus are bacilli or rod-shaped bacteria. Many bacilli occur singularly, but chains may
• also be observed. Bacilli very greatly in length and diameter. Staphylococcus aureus and Streptococcus pneumoniae are cocci or spherical bacteria. Cocci may occur singularly, in pairs (as in Streptococcus pneumoniae), or in clusters (as in Staphylococcus aureus). R. rubrum is a spirillum or curved bacterium, Spirilla always occur singularly.
• Differential Stains:
• Differential stains are more complex than simple ones and use more
• than one stain to differentiate cellular components. They are used to
• examine structural differences between bacterial groups or to provide
• contrast to different structures within the same organism.

This pertains to a parallel arrangement of rod-shaped cells. This characteristic also called picket fence arrangement is common to many corynebacteria

These are distinct reddish purple granules within cells that show up when the organisms are stained with methlene blue.They are masses of volutin, a polymetaphosphate

This pertains to irregularity of form, that is demonstarting several different shapes.

• Examples of dyes used are nigrosin and india ink. We use nigrosin.
• 1.) Organisms are dispersed into a small drop of nigrosin. Drop shoud not exceed 1/8 diameter an shoud be near the end of the slide.
• 2.) Spreader slide is moved toward drop of suspension untill it contacts the drop causing the liquid o spread along its spreading edge,
• 3.) Once sreader slide contacts the drop on botton slide the suspension will spread out along the spreading edge.
• 4.) Sprader slide is pushed to the left dragging the suspension over the bottom slide. After the slide has air-dryed, it may be examined under oil immersion
• SECOUND PROCEDURE USED
• 1,) A loopful of nigrosin is placed in the center of a clean microscope slide
• 2.) A steri inoculating wire is used to transfer the organism to the liquid and mix the organisms into the stain
• 3.) Suspensions of bacteria is spread evenly over an area of one or two centimeters with the straight wire.
• 4.) Once the preparation has completely air-dryed it can be examined under oil immersion

• Morphology: Coccus
• An average coccus is about 0.5-1.0 micrometer (µm) in diameter
• Arranged in grape like irregular clusters

• Morphology: Rod shaped, single
• Arrangement: Bacillus, one rod
• Size:An average bacillus is 0.5-1.0 µm wide by 1.0-4.0 µm long.

Negative stains can be useful in studying the morphology of bacterial cells and characterizing some of the external structures, and it is also useful in determing cell dimensions and also in observing spirochaetes. The disadvantage is that little or nothing is learned about the internal structure of the cell

• Crystal Violet: Stains gram positive bacteria purple
• Iodine: A mordant that complexes with the Crystal Violet and forms an insoluble complex in gram positive cells.
• Safranin: Counter stain used to dye gram negative bacteria after slide has been treated with acetone

• 1.) Cover the smear with Crystal Violet and let stand for 20 secs- 1 minute
• 2.) Briefly wash off the stain using distilled water, drain off excess water
• 3.) Cover the smear with Gram's Iodine solution and let stand for1 minute
• 4.) Wash off the grams iodine hold the slide a a 45-degree angle and allow 95% alcohl to flow down the surface of the slide.
• 5.) Stop decolorization by washing the slide with a gentle stream of water
• 6.) Cover the smear with safranin for 1 minute.
• 7.) Wash gently, blot dry,and air dry
• 8.) Examine slide under immersion oil

• Gram Negative

• Gram Positive

• Gram negative
• Morphology: Bacillus, rod
• Arrangement: clusters
• Size:3.0 to 5.0 µm long

• Gram Positive
• Morphology: Bacillus, rod
• Arrangement:Single
• Size: An average bacillus is 0.5-1.0 um wide by 1.0-4.0 um long

It is a critcal procedure in identifying an unknown bacteria

It helps in the identification of bacteria, which makes targeting a pathogen much easier

Endospores are very dehydrated structures that are not actively metabolizing. They are resistant to heat, radiation, acids, chemicals due to a protein coat they have called an exosporium.

• 1.) Cover smear with a small piece of paper toweling and saturate with Malachite green. Steam over boiled water for 10 minuets. Add more stain if stain boils off
• 2.) After slide has cooled sufficiently, remove the paper toweling and rinse with water for 30 ec.
• 3.) Counterstain with Safranin for about 1 minute.
• 4.) Rinse briefly with water to remove safranin
• 5.) Blot dry with bibulous paper examine slide under immersion oil

• 1.)Make a heavy suspension of bacteria by dispersing several loopfuls of bacteria in 5 drops of steril water
• 2.) Add 5 drops of carbolfuchsin to the bacterial suspension
• 3.)Heat the carbolfuchsin suspension of bacteria in a beaker of boiling water for 10 minutes
• 4.) Mix several loopfuls of bacteria in drop of nigrosin on the slide.
• 5.) Spread the nigrosin bacteria mixture on the slide.
• 6.) Allow the smear to air dry. Examine under oil immersion

Genera Bacillus and Clostridia

Bacteria in the genus Mycobacterium and some in the genus Nocardia contain a waxy material n their cell walls called mycolic acid

Carbolfuchsin. Counterstain is Methylene blue

• 1.) Cover smear with carbolfuchsin Steam over boiling water for 10 minutes Add additional stain if it boils over
• 2.) After slide has cooled, decolorize wiyth acid-alcohol for 15-20 seconds
• 3.) Stop decolorization action of acid-alcohol by briefly rinsing with water
• 4.) Counterstain with Methylene blue for 1 minute
• 5.) Rinse breifly with water to remove excess methylene blue
• 6.) Blot dry with bibulous paper Examine dirctly under oil immersion

Mycobacterium tuberculosis and Mycobacterium leprae

• Staph is blue because it is negative
• Myco is pink/purple because it is positive
• Mycobacterium smegmatis is a rod/ bacillus shape
• Staphylococcus aureus is coccus

Quadrant Streaking,

• NON MOTILE

Motile, posses flagella. Agar is Motility Medium Triphenyl Tetrazolium Chloride, a semi solid .4% agar with indicator dye

Motility Medium Triphenyl Tetrazolium Chloride

Proteus mirabilis

Staphylococcus aureus

Fluid Thioglycollate MediaResazurin reacts with oxygen and turns pink. Boil the media with sodium thioglycallate removes oxygen

This group can grow in the presence of oxygen and are not usually harmed by its presence. Their metabolism does not require oxygen

Facultative anaerobe

Obligate Aerobe

Obligate Anaerobe

Colony Formng Units

Escherichia coli

An organism that requires high amounts of salt concentrations

An organism that does not require high salt environments, but can live in them

Organisms that can grow in high amounts of sugar

Mannitol Salt Agar

• Escherichia coli
• Staphylococcus aureus
• Halobacterium salinarium
• Bacillus megaterium