Environmental Microbiology -Laboratory Manual-

IIsolation and PurificationUse of MicroscopeSimple StainEnvironmental Microbiology-Laboratory Manual-anindrya.pahlawan@ymail.comEnvironmental Management technologyFaculty of civil and environmental engineeringItb, 20101Exp. 1 - Aseptic Culture TransferExp. 2 Pure Culture Isolation TechniqueExp. 3 Isolation from Environment

Isolation and Purification2Why Isolating ?Microorganism is everywhereIsolation : microorganism population individual species for study purpose Pure Culture3Laboratory Apparatus (1)AutoclaveCulture TubePetri Dish

4Laboratory Apparatus (2)Inoculation NeedleBunsen BurnerIncubator

5MediumBrothSemi-SolidSolid

6Solid MediumDeep Tube AgarSlant AgarPlate Agar

7Making Media

8Aseptic Culture Transfer9Exp. 1 - Aseptic Culture TransferTransferring m.o. culture from a medium to another standard procedure in microbiology labM.o. are everywhere external contamination ASEPTIC TRANSFER :DisinfectionBunsen Burner10) Position the handle end of the wire in the light blue cone of the flameii) Draw the rest of the wire upwards slowly into the hottest region of the flame immediately above the blue cone.iii) Hold there until it is red hot.iv) Ensure the full length of the wire receives adequate heating.v) Allow to cool for a few seconds in the air, then use immediately.vi) Do not put the loop down, or wave it around.vii) Re-sterilize the loop immediately after use

Sterilizing Loop Using Bunsen burner11Inoculation

12Sterilizing Tube Mouth Using Bunsen Burner

13Transfer from Liquid Medium to Broth TubeIn tube to tube transfers by loop or straight wire, both the tube containing the inoculums source and the tube to be inoculated are usually in the hand at the same time.

14Transfer from Slant Agar to Another

15Transfer from Agar Media to Petri Dish

16Pure Culture Isolation17There is no individual species living by its ownLaboratory separated using pure culture to study biochemical, morphology, etcDeveloped by Robert KochColony m.o. mass growth seen on solid medium, each colony represents multiplication of singe organism transferred to agar mediumStreak plate, pour plate, spread plate18Streak Platethe dilution of bacteria by systematically streaking them over the agar surface in a petri dish to obtain isolated cells During incubation, the isolated microbes multiply Excessive moisture from the condensation of water, derived from the initial cooling of the hot sterile media. If this water drops down onto the agar surface, spreading and merging of colonies can occur one should INVERT the plates after streaking them and when they are incubated.

19Streaking Method

20After Incubation

21Pour platealso called the loop dilution methodinvolves the successive transfer (serial dilution) of bacteria from the original culture to a series of tubes of liquefied agarthe concentration of bacteria in the first tube is lower than the concentration in the original culturethe process is repeated for a third tube of liquefied agarthe contents of each tube is poured into a separate Petri platea major concern in preparing pour plates is the temperature of the agar

22Spread Plate

23Isolation from EnvironmentIsolation from AirIsolation from Soil24Colony Counter

25Using Microscopes

26Terms & DefinitionA microscope (from the Greek: mikrs, "small" and skopen, "to look" or "see") = is an instrument to see objects too small for the naked eye Microscopy the science of investigating small objects using such an instrumentMicroscopic invisible to the eye unless aided by a microscope.Optical, electron, etc.27Optical MicroscopeUsing visible wavelengths of lightthe simplest and most usedhave refractive glass to focus light into the eye or another light detectorTypical magnification of a light microscope is up to 1500 x with a theoretical resolution limit of around 0.2 micrometers or 200 nanometersCaution use of immersion oil in 100x magnificationCoarse focus and fine focus

28Components of A MicroscopeA microscope consists of non optic components & optic components

29Simple Stain30StainingBacteria extremely small m.o., transparantStaining is an auxiliary technique used in microscopy to enhance contrast in the microscopic image, highlighting structures in biological tissues for viewingBasic principle ion bonding of cellular component and active component of dyeSimple Stain & Differential StainAcid and base stainAcid : dye (-), chinese ink, NigrosinBase : dye (+), fuchsin carbol, methylene blue31Preparing SmearHeat fixation aims to attach m.o. to the slide, and to preserve the shape of the cells or tissue involved as much as possibleAfter placing several drops of water on the slide, smear the bacteria from the inoculating loop from the top to the bottom of the slide

32Stain

33Morphological Shapes of Bacteria

34SAFETY FIRST !`Work steril is the success of microbiology laboratory practice.35Gram Staining

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