Identifying and Identifying and Controlling MicrobesControlling Microbes

Unit 7Unit 7

Donna Howell

Medical Microbiology

Blacksburg High School

Body Fluids Body Fluids : : BloodBlood

•Blood can contain:– Hepatitis virus

(B & C)– HIV virus– Cytomegalovirus

•Can be infected by blood through cuts, abrasions, needles, onto mucous membranes

I don’t vant to suck your blood!

Body Fluids Body Fluids : : FecesFeces

Feces can contain:– Salmonella bacteria

– Shigella bacteria

– Rotavirus

– Hepatitis A virus

– Others

•Can be infected through dirty hands to mouth.

Body Fluids Body Fluids : : UrineUrine

•Urine can contain:– Cytomegalovirus

•Urine is normally a

sterile body fluid•Can be infected with

urine through hand

to mouth contact

Body Fluids Body Fluids : : Respiratory Respiratory SecretionsSecretions

Respiratory secretions can contain:

– Mononucleosis virus

– Cold virus

– Influenza (flu) virus

– Meningitis bacteria

– Many others

•Can be infected through hand to mouth, or through sneezing and coughing.

Body Fluids Body Fluids : : VomitusVomitus

Vomit can contain:– Any gastrointestinal

virus, (such as Rotavirus)

•Can be infected by hand to mouth contact.

Laboratory EquipmentLaboratory Equipment

Some equipment we use in the microbiology lab are:•Microscope•Petri dish & agar•Inoculating loop•Incubator

Pics of these are in your notes

MicroscopeMicroscope

We will be using two types of microscopes in this class:•The compound light microscope (to view bacteria, protists, and some fungi)•The dissecting microscope (to view larger specimens)

Compound Light Compound Light MicroscopeMicroscope

We will be using an oil immersion objective this year to view bacteria. This allows us to magnify a specimen 1000 times! To use it, you place a drop of oil onto the specimen on the slide, and swing the objective into place.

Petri DishPetri Dish

A shallow plastic flat-bottomed dish with a lid used to culture bacteria. Contains agar, which is a gelatin-like substance that has nutrients in it that bacteria require for growth.

Inoculating LoopInoculating Loop

This is used to transfer a specimen to the agar plate. Ours will be disposable for the most part, but can be metal. If we use the metal ones, we must sterilize it in the Bunsen burner before use.

IncubatorIncubatorUsed to incubate the inoculated Petri plates – encourages growth of bacteria. Most incubated at body temperature (37oC)

Pure CulturesPure CulturesWhen working with microorganisms, it is important to work with pure cultures, which are cultures composed of only 1 type of organism. You do not want other organisms contaminating your pure culture.

Plating BacteriaPlating BacteriaThere are two plating methods we will use in this class:•Smear method•Streak plate method

Smear MethodSmear Method

This is a method of putting a specimen an a Petri dish where you just rub the cotton swab over the whole agar surface. Used when you just want to grow ALL bacteria, perhaps to get a colony count.

Streak Plate MethodStreak Plate Method

The streak plate method is used when you are trying to isolate individual colonies (types) of bacteria.

Universal PrecautionsUniversal Precautions•Rules you follow in the medical field•Designed to minimize potential of contracting a disease•Overall premise: Treat ALL specimens as if they are contaminated with a deadly disease.

The “Rules” of Universal The “Rules” of Universal PrecautionsPrecautions

1. Treat all body fluids as if they were contaminated.

2. Use disposable non-latex gloves when exposure to body fluids is possible.

3. Wash hands thoroughly after gloves removed.

4. Use protective clothing / masks to prevent splashes.

5. Use these rules when culturing bacteria and cleaning up the lab.

Aseptic TechniqueAseptic Technique

This is a method that prevents the introduction of contaminants into your pure cultures. Remember: microbes are found in the air, on countertops, on your skin, etc. If you are not careful, you can introduce some of these into your pure cultures.

Aseptic TechniqueAseptic Technique

Aseptic technique involves the following:•Sterilize any instruments you are using to work with bacteria.•Always keep the lid on your agar plate unless working with the cultures.•Do not set test tube lids on counters, etc.•Use sterile gloves.•Other

Streak Plate MethodStreak Plate Method1. With inoculating

loop, transfer specimen to first quadrant of agar plate. Flame loop.

2. Rotate plate, streak second quadrant. Flame loop.

3. Repeat step #2 to streak the 3rd and 4th quadrants.

4. Be very gentle – agar is soft, and you don’t want to dig around in it!

Classifying and Classifying and Identifying Identifying

Because a lot of microorganisms look alike under the microscope, we need methods other than just looks to classify them. The next few slides talk about ways we do this!

Differential StainingDifferential Staining

•A procedure that takes advantage of the physical and chemical properties of different groups of bacteria.•Allows us to differentiate between bacteria with differing cell walls.

Differential StainingDifferential Staining

Organisms can be stained with many different types of stains. For instance, the Gram stain places bacteria into one of two main groups: Gram +, or Gram -.

The Gram StainThe Gram Stain

•Developed in 1884 by Mr. Gram, a Danish physician.•Divides most bacteria into one of two groups: Gram-positive and Gram-negative.

The Gram StainThe Gram Stain

Gram-positive: Organisms appear purple because their cell wall retains the purple dye. This is due to a cell wall with a high polysaccharide content, and low lipid content.

The Gram StainThe Gram Stain

Gram-negative:Organisms appear red because their cell wall does not retain the purple dye; instead, it retains the red dye. This is due to a cell wall with a high lipid content, and low polysaccharide content.

Gram Stain ProcedureGram Stain Procedure1. Heat-fix bacterial smear on slide.2. Flood slide with crystal violet stain, let sit for 1 minute. Rinse until clear.3. Flood slide with iodine, let sit for 1 minute, rinse until clear.4. Drip alcohol over slide quickly, rinse good.5. Flood slide with safranin stain, let sit for 1 minute. Rinse until clear.6. Dry and observe under microscope.

Morphological Morphological CharacteristicsCharacteristics

This means looking at how the organism looks – distinguishing characteristics.

How are the cells shaped? Do they have flagella? Spores?

Biochemical TestsBiochemical Tests

We can place bacteria into different chemicals, and see what they do to that chemical. For instance, if placed in lactose, do they ferment it? If in nitrogen, can they “fix” it? Can they oxidize sulfur?

DNA FingerprintingDNA Fingerprinting

We can look at the DNA code of the organism. This is useful to match species, and to trace ancestry.

Isn’t Micro the Isn’t Micro the