Microbiology -SELF CHECKSUBMITTED BY: Charlene P.

Binuya

SUBMITTED TO: Prof. Gladys Aquino

July 19, 2012

I. Introduction to Microbiology Lecture: Bacteriology- the study of bacteria. Bacteria- one of the three domain in 3 domain system of classification; members of this domain are prokaryotes; the other 2 doamins are archeae and eucarya. pathogenecity- the ability to cause disease. virulence- a measure of pathogenecity some pathogens are more or less virulent than others. 2. Historical DevelopmentBacteria and protozoa were the first microoraganisms to be observed by humans. Anton Van leeuwenhoek (1632-1723)- first person to see live bacteria and protozoa. Louis Pasteur (1822-1895)- he developed a process to kill microbes that were causing wine to spoil an economic concern to Frances wine industry. Pasteurization-can be used to kill pathogens in many types of liquids. Robert Koch (1843-1910)- he discovered the bacterium (mycobacterium tuberculosis) that causes tuberculosis and the bacterium (vibrio cholerae) that causes cholera. 3. Divisions of Microbiology

Laboratory: 1. Expectations/ requirements Microbiology Laboratory: Spectrophotometer, Quebec colony counter, Balances,Homogenizer,pH meter,Hot plat Vortex shaker, Magnetic stirrer, Membrane filter assembly, Compound Microscope,Water bath, Hot air oven, Autoclave or pressure cooker,Incubator, Refrigerator, Centrifuge, Water distillation assembly 2. Rules to follow in handling, processing, disposal of specimens/waste in Microbiology -handle all specimen as if infectious. -all technical staff should follow the universal and other biosafety precausions while handling and disposing the microbiology specimens. -specimen disposal: develop policy for disposal of medical waste: establish and follow disinfection procedures,comply with local regulations, include policy of disposal of rejected specimens.

3. Barrier precautions Infection control is a general term referring to any method or device used to prevent contact with potentially infectious body fluids, including facial masks, doubled gloves and fluid resistant gowns. 4. Proper use of different apparatus, materials and equipment: Petri dish- a swallow , circular container made of thin glass or clear plastic, with a loosely fitting, overlappinf cover;used in microbiology laboratories for cultivation of microorganisms on solid media. Inoculating loop- is a simple tool used mainly by microbiologists to retrieve an inoculum from a culture of microorganisms. The loop is used in the cultivation of microbes on plates by transferring inoculum for streaking. Inoculating needle- An inoculating needle is a device used in Microbiological experiments. It is a needle made up of nichrome wire with a handle at the other end. It is used to tease fungal hyphae on a slide. 5. Review use and care of microscope - Always use two hands to move the microscope. Place one hand around the arm, lift the scope, and then put your other hand under the base of the scope for support. - Always observe the specimen or object using the lowest power object first. - Focus using the coarse adjustment knob to bring the object into focus. Bring the object into sharp focus by using the fine adjustment knob. - Focus, and then move to a higher power objective, if needed. -Use only the FINE ADJUSTMENT KNOB when using the HIGHEST (longest) POWER OBJECTIVE. -Keep both eyes open to reduce eyestrain. Keep eye slightly above the eyepiece to reduce eyelash interference. -To find out the total magnification of the object, multiply the power of the eyepiece lens (10X) by the power of the objective. II. Laboratory safety and infection control Lecture: sterilization- the destruction of all microorganisms in or on something. autoclave- an apparatus used for sterilization by steam under pressure.

direct heat- Using a flame to sterilize a needle or other body modification implement is generally considered unacceptable. There is little real world data available on the effectiveness of direct flame in killing microbial life, and when it comes to items that break the skin, the only context in which flaming is still used is in cases where a needle has to be reused immediately and there are no other options. oven- Dry heat is produced by a hot air oven. Glassware, glasssyringes, forceps, scalpel, pipettes, flasks, petri dishes, etc. are sterilized in an oven. incineration- process of killing the microorganisms by using flame (heat), hence also called flame sterilization. Pasteurization- a heating process that kills pathogens in milk, wines and other beverages. boiling- Boiling water is 100 degrees Celsius. Although this will theoretically kill most organisms other than endospores and some viruses in half an hour, because endospores are not killed and the consistency is poor, no health boards consider boiling as a viable form of sterilization. disinfection- the act of disinfecting, using specialized cleansing techniques that destroy or prevent growth of organisms capable of infection. Chemicals: alcohol- Ethanol or iso-propanol (70%) is used to sterilize the working tabletop, inoculation chamber, etc. chlorine- It is a disinfectant, decolorant, and irritant poison, and is used for disinfecting, fumigating, and bleaching. Barrier precautions Infection control is a general term referring to any method or device used to prevent contact with potentially infectious body fluids, including facial masks, doubled gloves and fluid resistant gowns. Control of nosocomial infections Laboratory: 1. sterilization- the destruction of all microorganisms in or on something. autoclaving- An autoclave is generally considered to be the only form of sterilization appropriate for a body modification studio, although some low-volume home studios may use chemical sterilization. It should be noted that cleaning is the most important part of sterilization - unclean tools may not sterilize properly.

direct heating- Using a flame to sterilize a needle or other body modification implement is generally considered unacceptable. There is little real world data available on the effectiveness of direct flame in killing microbial life, and when it comes to items that break the skin, the only context in which flaming is still used is in cases where a needle has to be reused immediately and there are no other options. filtration2. cleaning of working area with Clorox and other chemicals It is far a more powerful anti-microbial agent than alcohol. Drug treatment clinics regularly advise addicts to immerse their syringes in a thinned down bleach solution. However, they are starting to move away from this because the bleach kits often don't kill Hepatitis, and sometimes don't even kill the AIDS virus. III. Bacterial structure and function, growth and nutrition Lecture: 1. Characteristics of bacteria according to: Physiology: structure, function A unicellular organism size of 0.5-2.0 micrometer, prokaryotic cell, for production they multiply using binary fission, both DNA and RNA and the smallest hemophilus and largest bacillus anthracis. Bacteria multiply by cell division that results in the production of two daughter cell from one parent cell. Bacterial morphology There are three basic morphologies of bacteria (based on the shape of a single cell): bacillus (little rod), coccus (grain or berry) or spirillum (coiled or helical). Classificaton It seeks to describe the diversity of bacterial species by naming and grouping organisms based on similarities. Bacteria can be classified on the basis of cell structure, cellular metabolism or on differences in cell components such as DNA, fatty acids, pigments, antigens and quinones. While these schemes allowed the identification and classification of bacterial strains, it was unclear whether these differences represented variation between distinct species or between strains of the same species.

Growth requirements: Different environmental factors: nutrients and moisture, temperature, ph, osmotic pressure and composition of the atmosphere. 2. Bacterial growth curve four phases of growth curve: lag phase, log phase, stationary phase and death phase. 3. Bacterial identification: Microscopy: morphology bacteria vary greatly in size, usually ranging from spheres measuring about 0.2 um in diameter to 10.0 um long spiral shaped bacteria. cultural/colony characteristics colony morphology in cludes the size, color, overall shape, elevation and the appearance of the edge or margin of the colony. 4. Quality assurance- Quality control Laboratory: Role of microscopy in bacterial identification: bright field Principle: light pass through the specimen and then through a series of lenses that reflect the light that result in magnification of the organisms present in the specimen. Magnification: ocular lens X objective lens Resolution: extent to which detail in the magnified object is maintained. Contrast- achieved by staining techniques to allow them to be differentiated from background material and debris. phase contrast Principle : Beams of light pass through the specimen and are partially deflected by the different densities or thickness (refractive indices) of the microbial cells or cell structures. The greater the refractive index of an object, the more the beam of light is slowed, which results in decreased light intensity.

electron Uses electron instead of light to visualized objects, and instead of lenses, the electrons are focused by electromagnetic fields and form an image on a fluorescent screen, like a television screen. Allows magnification in excess of100,000X dark field Uses special type of condenser that allows light to deflect as they pass through the specimen which make the background dark and the specimen to be visualized bright. Effective for live unstained specimen for culture fluorescent Uses fluors or fluorochrome Principle: Certain dyes, called fluors can be raised to a higher energy level after absorbing ultraviolet light. When the dye molecules return to their normal, lower energy state, they release excess energy in the form of visible light. Preparation of Bacretial smear for staining from: directly from specimen (throat swab or any swab) Placing the bacterial sample on the slide Place a drop of water into the wax circle that has been created on the slide. Using a sterilized and cooled inoculation loop, obtain a very small sample of a bacterial colony. Gently mix the bacteria into the water drop. Staining techniques: simple stain- a single dye that is used to stain objects, enabling scientists to gain information about the objects. differential stain: bacterial staining procedures that enable differentiation of two groups of bacteria. gram stain- method for the differential staining of bacteria by treatment with Grams solution after staining with a triphenyl methane dye called also Grams method. acid fast stain- should also be used to detect Cryptosporidium species and Mycobacteria special stain- technique highlights the flagella of bacteria

IV. Bacterial genetics Lecture: 1. Basic concepts: Bacterial DNA and RNA a cell can mutate as result of accidental changes in its genetic material the DNA that makes up the genes of its chromosomes and thus can become better or less suited to its environment. Plasmids and Microbial resistance Plasmid is a small circular molecule of double- stranded DNA. It is refferd to as extrachromosomal DNA because it is not part of the chromosome. it found in most bacteria. DNA exchange in bacteria: transformation- involves the uptake of naked DNA transduction- involves bacteriophages and the acquisition of new bacterial genes. conjugation- involves the transfer of genetic information from one cell to another through a hollow sex pilus. V. Pathogenesis and Epidemiology Lecture 1. Basic concepts Pathogenicity is the ability to cause disease and pathogenesis is the step or mechanisms involved in the development of a disease. 2, Mode of transmission direct skin to skin contact, direct mucous membrane, airbourne, contamination of food and water by fecal material, arthropod vectors and indirectly by transfusion of contaminated blood or from an ill person or using nonsterilized needles. 3. Factors associated with pathogenecity and virulence: toxins- a poisonous substance produced by a microorganism. enzymes- a protein molecules that catalyzes a chemical reaction. capsules- an organized layer of glycocalyx, a firmly attached to the outer surface of a bacterial cell wall.

VI. Normal Flora Lecture: 1. Basic Concepts A persons indigenous microflora or indigenous microbiota (sometimes referred to as normal flora) includes all the microbes that reside on and within that person. 2. Body Sites with normal flora Areas that normal flora reside: skin,eyes,ears, upper respiratory tract, gastrointestinal tract and genitourinary tract. 3. predominant flora gastrointestinal tract 4. beneficial and harmful effects of normal flora -Human derive many benefits from their indigenous microflora,some of which have already been mentioned. some nutrients particularly vitamins K and B12, pantothemic acid, pyridoxine and biotin are obtained from secretions of certain intestinal bacteria. -source of irritants and antigens to stimulate the immune system. -respond to antibodies which enhance body protection against pathogens.

REFERENCES: Burtons Microbiology for the health sciences eight edition by Paul G. Engelkirk and Gwendolyn R.W. Burton