Lab 1

Media and media preparation

Important equipment in the lab Microscope Incubator : bacteria usually at 37c , fungi from 25 -30c Autoclave Safety Cabinet

Media in Microbiology Culture media contains the nutrients needed to allow microbe to grow. Types of media - General - Selective - Differential - Selective & differential Forms of media - broth - solid - semi-solid

How is media made? When lab personnel make media they measure out a quantity of dry powdered nutrient media, then add water They dispense the media into bottles (flask,tube), cap it and autoclave. The autoclave exposes the media to high temperature (121°C) and pressure (15 psi) for 20 minutes. Once the media is autoclaved it is sterile (all microoranism forms killed)

How is media made? 1- Suspend x g agar powder in x liter of distilled water. - measure required amount by using balance - Then add the powder to a flask - Add the right amount of d. water 2- Bring to the boil to dissolve completely 3- autoclaving 4- pouring

Safety To disinfect bench surfaces , we use Alcohol 70% Don’t eat or drink in lab Always wear gloves Clean bench after use Wash hands before leaving the lab

Lab 2

Condenser Lens: The function of the condenser lens is to collect the light from the illuminator and focus it on the specimen. Diaphragm or Iris: The diaphragm is used to control the amount of light reaching the specimen. In a student scope it is a rotating disk under the stage and above the condenser. Rules for Using the Microscope 1. Use two hands to carry a microscope, one hand holding the arm, the other holding the base. 2. Only use lens paper and water to clean the lenses.

Magnification : the ability to make small objects seem larger microscope has 4 magnifications: Scanning, Low , High & oil immersion. Each objective will have written the magnification. In addition to this, the ocular lens (eyepiece) has a magnification. The total magnification is the ocular x objective. Resolution: the ability to see fine details. Contrast: the ratio between the dark and the light. (difference in color)   Magnification Ocular lens Total Magnification Scanning 4x 10x 40x Low Power 100x High Power 400x Oil immersion 1000x

Numerical Aperture NA NA means The light-gathering ability of a microscope objective. The higher the NA value the better the resolution. For 100x lens , we usually add oil between the lens and slide. why ? - This is because that the refractive index of air is 1 and oil is ~ 1.5. So, NA value increases with oil.

Lab 3

Staining Simple staining : only one dye . Ex. Simple stain methylene blue Differential staining: more than one dye . Differentiation among bacteria is possible Ex. Gram stain Special staining: stain Special structures Ex. Capsule stain , spore stain.

Gram stain Hans Christian Gram , Danish scientist Classify Bacteria into Gram negative & Gram positive . Thin cell wall Thick cell wall

4 dyes 1- primary stain: ( crystal violet ) 1 minute 2- mordant : ( iodine ) 1 minute 3- decolorizer : ( alcohol or acetone ) 1-2 seconds 4- counterstain : ( safranin ) 1 minute

Lab 4

Parasites multicellular unicellular

Protozoa examples Trypanosoma spp. - flagellated protozoa - causes sleeping sickness disease

Protozoa examples Plasmodium falciparum - causes Malaria - blood smear

Helminth: nematodes (roundworm) Enterobius vermicularis worm egg

Helminth: nematodes (roundworm) Ascaris lumbricoides egg

Helminths: trematodes ( Fluke) Schistosoma Terminal spine lateral spine rudimentary lateral spine

Helminth: cestode (tapeworm) Taenia saginata (beef tapeworm) worm egg

Fungi Unicellular or multicellular eukaryotes.

Yeast examples Candida albicans Pseudohypae, hyphae and yeast

Mold examples Aspergillus spp. - septated hyphae

Rhizopus spp. - aseptataed hyphae sporangium spores

Lab 5 &6

Important terms colony - is a visible mass of microorganisms all originating from a single mother cell, therefore a colony constitutes a clone of bacteria all genetically alike.

Culture vs. subculture Culture: is a method of multiplying microbial organisms by letting them reproduce in predetermined culture media under controlled laboratory conditions Sub-culture: a new cell or microbiological culture made by transferring some or all cells from a previous culture to fresh growth medium.

Mixed vs. pure culture Mixed culture: two or more microgranism on the same plate.

Pure culture : only one microorganism on the plate

colony morphology Form : circular , filamentous Pigmentation : colors Texture : dry , moist , mucoid Surface : smooth, rough, shiny Size : tiny , large

Examples of colonies Zone of hemolysis on blood agar

Examples of colonies Swarming on plate ex. Proteus spp.

Examples of colonies Mucoid colony ex. Klebsiella spp.

Examples of colonies Irregular colony form ex. Bacillus

Streaking purpose :To have isolated colonies

Lab 7

The Vegetative Cell Gives Rise to One Spore Bacterial Cell Bacterial Cell Spore Bacterial Cell The endospore is able to survive for long periods of time until environmental conditions again become favorable for growth. The endospore then germinates, producing a single vegetative bacterium.

Not all bacterial species can form spores A few genera of bacteria produce Endospore such as Clostridium and Bacillus (Endospore production is associated with Gram Positive bacteria)

Schaeffer-Fulton Stain Procedure 1. Prepare a smear. Air Dry. Heat fix 2. Put the slide on steam rack 3. Flood the smear with Malachite Green stain 4. Steam slide for 5 minutes (be sure that the satin is not dry, if so , add more satin ) 5. Drain slide and rinse with DI water. 7. Flood smear with Safranin (counter stain). This stains the vegetative cell. (Leave for 1 minute) 8. Drain the slide and rinse with DI water 9. Dry 10. View slide under 100x oil immersion.

Endospore Stain Example Spores: Green Cell: Red or Pink Practical: Each student will make smear of Bacillus subtilis

Lab 8

Bacterial count

Bacterial count Total count is a count of cells including dead and live cells. Viable count is a count of only those cells that are alive in the sample. Also know as “viable cell counts” Concentrated samples are diluted by serial dilution The diluted samples can be either plated by spread plating or by pour plating

colony-forming unit CFU - a colony-forming unit is a unit used to estimate the number of viable bacteria cells in a sample

Procedure: - aseptically dilute sample - make 1:10 dilutions as instructed, based on sample - aseptically transfer 100 ul volume of dilutions that are to be plated onto agar plates - Spread the sample on the agar very well - incubate as instructed for growth of colonies - during next lab period, count colonies on plates - use the formula: cfu/ml = # of colonies x dilution amount plated ( ml)

Calculation Example Bacterial sample is diluted 1:10 (i.e. 1 ml diluted with 9 ml saline) 0.01 ml of 1/10 dilution is inoculated onto AC plates 87 colonies grow The Concentration of bacteria is cfu/ml = # of colonies x dilution amount plated ( ml) 87 x 10 .01 = 87,000 cfu/ml