1 Dr. Paul KONKUK UNIVERSITY DEPT. OF ENVIRONMENTAL ENGINEERING INTRODUCTION TO MICROBIOLOGY LABORATORY

2 INTRODUCTION TO MICROBIOLOGY LABORATORY

3  Everyone must wear a lab coat or lab apron while in the laboratory.  Do not work with an uncovered open cut.  Keep all sources of possible contamination out of your mouth--hands, pencils, laboratory ware, other items.  Do not smoke or eat in the laboratory.  Spills of materials containing viable organisms should be immediately contained with dry paper towels. The dry towel will soak up the spill and can then be sterilized. Following this, the area of the spill should be disinfected with bench disinfectant. Lab Safety LABORATORY RULES AND PROCEDURES:

4  Report accidents, such as a spilled culture or a cut, to the laboratory instructor. Our interest is safety.  Shoes must be worn at all times in the laboratory.  Observe aseptic technique at all times when dealing with microbial cultures.  Wash hands with soap and water or disinfectant before leaving the laboratory.

5 INSTRUMENTS & APPARATUSES

6 Microscope microscope is an instrument for viewing objects that are too small to be seen by the naked or unaided eye.

7 Microscopes allow magnified images of illuminated specimens to be viewed using 2 lenses (an objective and an eyepiece lens). Microscopes that use two lenses are called compound microscopes.

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9 TYPES OF MICROSCOPES Upright microscopes are used for viewing slide glass preparations. Inverted microscopes are used for viewing petri dishes or culture containers. Upright microscope Inverted microscope Stereoscopic microscopes These microscopes show the specimen three dimensionally Stereoscopic microscopes

10 Microscopy imaging techniques The most commonly used microscopy imaging technique is brightfield microscopy, where light is either passed through or reflected off a specimen. However, there are also techniques known as darkfield microscopy, phase contrast microscopy, differential interference contrast (DIC) microscopy, fluorescence microscopy, and polarizing microscopy. Each of these methods is best suited for different uses.

11 Brightfield microscopy Observation is made by viewing the light passed through or reflected off a specimen. Main uses: Viewing stained specimens Pathological exams Blood tests Wafer inspections

12 Darkfield microscopy A special condenser lens is used to illuminate the specimen diagonally, then observe light scattering off it. The field of view is darker than brightfield microscopy because illumination light does not enter the objective lens. Main uses: Microbiological imaging Blood tests

13 Phase contrast microscopy The optical phenomena of diffraction and interference are used to add light/dark contrast to a transparent specimen for imaging. There is no need to stain the specimen as in brightfield microscopy, so live specimens can be used. Bacilli in pond water with unidentified algae Under bright-field the bacteria would be exceedingly difficult to see in this particular situation, yet phase contrast reveals them with ease

14 Fluorescence microscopy Specimen is excited with a specific wavelength of light, then fluorescent emanations are observed.

15 PETRI DISH INOCULATION LOOP

16 Bunsen burner FLAMING ALOOP

17 INCUBATOR

18 In microbiology, an incubator is a device for controlling the temperature, humidity, and other conditions in which a microbiological culture is being grown. The simplest incubators are insulated boxes with an adjustable heater, INCUBATOR

19 As for temperature, most commonly used is approximately 36 to 37 degrees Celsius. Most bacteria, especially the frequently used E. Coli, grow well under such conditions. For other experimental organisms, such as the budding yeast Saccharomyces cerevisiae, a growth temperature of 30 °C is optimal.

AUTOCLAVE

HOT AIR OVEN

Laminar flow cabinet

Cell Counts by Hemocytometer

 9 large squares – 1mm x 1mm  Center square divided into 25 smaller squares - 0.2mm x 0.2mm = 0.04 mm 2  Depth of the counting chamber – 0.1 mm  Volume of a small square – 0.04 mm 2 x 0.1 mm = mm 3  1 ml = 1 cm 3 = 1000 mm 3 (10 mm = 1 cm)

25 Average # of cells in a small square X mm 3 Average # of cells in a small square X 25 X 101mm 3 Average # of cells in a small square X 25 X 10 X ml

40x magnification 1 mm

100x magnification 0.2 mm

400x magnification