Introduction LAB to Practical Microbiology PDF

‫المعهد التكنولوجي‬ ‫العالي للعلوم الصحية‬ ‫التطبيقية ببني سويف‬ Introduction LAB to practical Microbiology Prepared By: Amira Aly Supervisor: Dr. Manal Mohamed Abdelalim ...

‫المعهد التكنولوجي‬ ‫العالي للعلوم الصحية‬ ‫التطبيقية ببني سويف‬ Introduction LAB to practical Microbiology Prepared By: Amira Aly Supervisor: Dr. Manal Mohamed Abdelalim  Practical Outline ◉ How to use microscope (fresh examination of Yeasts / Saccharomyces) ◉ Media preparation (Nutrient Agar – PDA) ◉ Isolation of bacteria from different sources (Soil – Water – Skin – Hair – Saliva) ◉ Staining of bacteria (Simple Stain) ◉ Gram staining (+Ve & -Ve) ◉ Bacterial Enzymatic Activity (Biochemical tests including Starch hydrolysis test – Catalase test) ◉ Effect of antibiotics on bacterial cultures ◉ Systematic position of Fungal species of kingdom mycota 2 Laboratory Equipment 3  Incubator Autoclave Petri Dishes Inoculating Loop 4 Essentials for Microbiology Lab 5  Precautions to be taken in microbiology lab ◉ Treat all microorganisms as potential pathogens. ◉ Sterilize equipment and materials. ◉ Disinfect work areas before and after use. ◉ Wear protection & Wash your hands before and after wearing gloves. ◉ Do not eat or drink in the lab, nor store food in areas where microorganisms are stored. ◉ Label everything clearly. ◉ Autoclave or disinfect all waste material. 6  Thanks! ANY QUESTIONS? 7  ‫المعهد التكنولوجي‬ ‫العالي للعلوم الصحية‬ ‫التطبيقية ببني سويف‬ How to use Microscope / Yeast Examination Under Light Microscope Prepared By: Amira Aly Supervisor: Dr. Manal Mohamed Abdelalim  How to use microscope? ◉ Learn the microscope components ◉ Place the microscope on a clean and flat surface ◉ Use the prepared slide to begin with it and place the slide on the stage then turn on the microscope. ◉ Adjust the eyepiece, if the microscope is binocular, leave both eyes open. ◉ Follow the low to high rule and use the adjustment knobs to focus the slide. ◉ After the examination is done, you have to clean the microscope lens using lens paper or xylene. 2  What is light microscope? ◉ Light microscope is a laboratory instrument which uses the visible light in order to detect and magnify small objects ◉ Microscopes are made up of lenses for magnification, each with its own magnification powers. Depending on the type of lens, it will magnify the specimen according to its focal strength. ◉ Its function depends on its ability to focus a beam of light through a specimen – which should be transparent- in order to allow easy and quick penetration of light and finally producing an image with suitable and good magnification and resolution. ◉ There are two types of light microscope depending on the No. of lenses: simple light microscope and compound light microscope. 3 Principle of Light Microscope 4  Types of Light microscope ◉ Bright-field light microscope ◉ Phase contrast light microscope ◉ Dark-Field light microscope ◉ Fluorescence light microscope 5  Light Microscope Components There are three structural parts of the microscope i.e. head, base, and arm. Head ◉ Head – This is also known as the body. It carries the optical parts in the upper part of the microscope. Arm ◉ Arm– This is the part connecting the base and to the head and the eyepiece tube to the base of the microscope. It gives support to the head of the microscope and it is also used when carrying the microscope. Base ◉ Base – It acts as microscopes support. It also carries microscopic illuminators. 6  Role of each part ◉ Eyepiece – also known as the ocular. This is the part used to look through the microscope. It’s Eyepiece found at the top of the microscope. Its standard Eyepiece tube Head magnification is 10x with an optional eyepiece Nose Piece having magnifications from 5X to 30X. Objective lenses ◉ Eyepiece tube – it’s the eyepiece holder. It carries the eyepiece just above the objective lens. ◉ Nose piece – It holds the objective lenses. It is movable hence it can revolve the objective lenses depending on the magnification power of the lens. ◉ Objective lenses – These are the major lenses used for specimen visualization. They have a magnification power of 4x-100X. There are about 1- 4 objective lenses placed on one microscope. Each lens has its own magnification 7 power.  To be Continued… ◉ The Adjustment knobs – These are knobs that are used to focus the microscope. There are two types of adjustment knobs i.e fine adjustment knobs and coarse adjustment knobs. ◉ Stage – This is the section in which the specimen is placed for viewing. They have stage clips that Stage Clips hold the specimen slides in place. The most Mechanical Stage Aperture Coarse Adjustment common stage is the mechanical stage, which Fine Adjustment allows the control of the slides by moving the Stage Controls slides using the mechanical knobs on the stage instead of moving them manually. ◉ Aperture – This is a hole on the microscope stage, through which the transmitted light from the source reaches the stage. 8 ◉ Microscopic illuminator – This is the microscopes light source, located at the base. It is used instead of a mirror. It captures light from an external source of a low voltage of about 100v. ◉ Condenser – These are lenses that are used to collect and focus light from the illuminator into the specimen. They play a Condenser major role in ensuring clear sharp images are Microscopic illuminator produced with a high magnification of 400X and above. 9 10  Yeast (Saccharomyces) ◉ Yeasts are eukaryotic, single-celled microorganisms classified as members of the fungus kingdom. ◉ Saccharomyces cerevisiae or baker’s yeast is the budding yeast used for bread-making, where the carbon dioxide produced by growth in the dough causes the bread to rise. Essentially similar yeasts, but now given different species names, are used for production of beers, wines and other alcoholic drinks. 11  Practical Part for Using Light Microscope ◉ First, rehydrate yeast powder or grains by adding suitable amount of distilled water ◉ Place Few drops of Safranin stain for morphology and structure visualization ◉ Place a fine drop on a clean slide and place the cover over it. ◉ Place prepared slide on microscopic stage and start examination. 12 Saccharomyces cerevisiae under the microscope in Lab. 13  Thanks! ANY QUESTIONS? 14  ‫المعهد التكنولوجي‬ ‫العالي للعلوم الصحية‬ ‫التطبيقية ببني سويف‬ Media preparation Prepared By: Amira Aly Supervisor: Dr. Manal Mohamed Abelalim  Questions for the previous section ◉ What is the total magnification power of light microscope if you are using the 40X objective lens? Note that: Ocular lens magnification power is 10X ◉ What is the role of Adjustment knobs? 2  Media Preparation ◉ Media preparation is the process of mixing nutrients, agents for buffering and maintaining the osmotic balance, as well as selective inhibitors or indicators to create an agar or broth that supports the growth and the differentiation of either bacteria or fungi. 3 Tools and instruments that we are going to use! 4  Nutrient agar ◉ Nutrient Agar is used as a general purpose medium for the cultivation of less fastidious microorganisms, can be enriched with blood or other biological fluids. ◉ Agar is a solidifying agent 5  How to prepare nutrient agar? ◉ Measure and Suspend 28g of nutrient agar powder in 1L (1000 ml) of distilled water. ◉ Mix and heat to boiling to dissolve the medium completely. ◉ Sterilize by autoclaving at 121°C for 15 minutes. ◉ Pour the liquid into the petri dish and wait for the medium to solidify. Be sure that you are preparing the agar in the clean environment to prevent any contamination. 6  PDA agar ◉ Potato dextrose agar (PDA) contains dextrose as a carbohydrate source which serves as a growth stimulant and potato infusion that provides a nutrient base for growth of most fungi. Agar is added as the solidifying agent. 7  How to prepare PDA agar? ◉ Measure and suspend 39 grams of PDA agar (4 gm potato starch, 20 gm dextrose, 15 gm agar) in 1L (1000 ml) distilled water. ◉ Mix and heat to boiling to dissolve the medium completely. ◉ Sterilize by autoclaving at 121°C for 15 minutes. ◉ In specific work, when pH 3.5 is required, the medium should be acidified with sterile 10% NOTE: Acid or antibiotic is added to inhibit the tartaric acid. The amount of acid required for bacterial growth. A specified amount of sterile tartaric 100 ml. of sterile, cooled medium is acid (10%) may be incorporated to lower the pH of the medium to 3.5 so that bacterial growth is approximately 1 ml. Do not heat the medium inhibited. after the addition of the acid. 8 How to pour media on petri dish? 9  Storage of petri dishes ◉ Once you prepare the nutrient agar in the petri dish, store them at 2-8°C. ◉ Agar plates petri-dishes should be stored upside down to prevent condensation. 10  Thanks! ANY QUESTIONS? 11  ‫المعهد التكنولوجي‬ ‫العالي للعلوم الصحية‬ ‫التطبيقية ببني سويف‬ Isolation of Bacteria from Different Sources Prepared By: Amira Aly Supervisor: Dr. Manal Mohamed Abdelalim  Questions From the previous section ◉ What are the steps to prepare nutrient agar? ◉ What is the difference between Nutrient Agar and Potato Dextrose Agar? 2  Storage of petri dishes ◉ Once you prepare the nutrient agar in the petri dish, store them at 2-8°C. ◉ Agar plates petri-dishes should be stored upside down to prevent condensation. 3 How to pour media on petri dish? 4  Bacterial Isolation Isolation of bacteria forms a very significant step in the diagnosis and management of the illness. There are two main ways to isolate organisms. ◉ Streaking for isolation on an agar plate ◉ The pour plate method 5  Bacterial isolation from soil ◉ The soil sample is collected. ◉ After that, the soil sample is dissolved in distilled water. then serial dilution is performed. ◉ Then the Streak plate method is performed on the sample with the highest dilution factor. ◉ The spread plate is incubated at an optimum temperature (based on the type of microorganism you want to isolate). ◉ After incubation microbial colony will form of your desired microorganisms. 6 Quadrant Streak method 7  Bacterial isolation from Air ◉ The Isolation of Microorganism From Air is performed by using the settle- plate technique. ◉ In this method a suitable medium is poured over a sterile petri dish and then allow it to solidify. After that the plate is exposed to the open air for a few minutes (5 mins – 10 mins). ◉ Then the plates are incubated for the formation of microbial colonies on the plate. 8  Growing Bacteria from Hair in Nutrient Agar Simply by placing a hair strand on petri dish and incubate it for 24 h at 37°c. 9  Swabbing a petri dish using different sources Take a clean cotton laboratory swabs dipped into saline and roll it on your skin, phone, lab note or any other choice and then Spread it on the petri dish. 10 Growing Fungi in petri dish 11  Note that, ◉ Do NOT forget to label you Petri dish by (type of medium – your initials – Date – bacteria source of isolation) ◉ Incubate Nutrient Agar petri dishes for bacteria at 37°c for 24 h or 48 h. ◉ Incubate Potato dextrose agar at 25°c – 27 °c for 7 days. 12  Thanks! ANY QUESTIONS? 13  ‫المعهد التكنولوجي‬ ‫العالي للعلوم الصحية‬ ‫التطبيقية ببني سويف‬ Simple Staining Prepared By: Amira Aly Supervisor: Dr. Manal Mohamed Abdelalim Bacterial Growth on Nutrient Agar Colony 2  Simple Staining ◉ Simple staining involves directly staining the bacterial cell with a positively charged dye in order to see bacterial detail. ◉ The simple stain can be used as a quick and easy way to determine the cell shape, size, and arrangements of bacteria. True to its name, the simple stain is a very simple staining procedure involving a single solution of stain. Any basic dye such as methylene blue, safranin, or crystal violet can be used to color the bacterial cells. 3 Summary of Simple Staining Steps 4  Preparation of Bacterial Smear 1. Draw a circle on clean and grease free microscopic slide and flip it over in order the circle to be on bottom 2. Add a drop of dist.H2O inside the circle of slide. 3. Using a sterilized inoculating loop to transfer an isolated colony from a culture plate to a slide with the water drop. 5  Preparation of Bacterial Smear 4. Disperse the bacteria on the loop in the drop of water on the slide and spread the drop over an area the size of a dime. It should be a thin, even smear. 5. Allow the smear to dry thoroughly until you see thin white haze. 6. Heat-fix the smear cautiously by passing the underside of the slide through the burner flame two or three times. It fixes the cell in the slide. Do not overheat the slide as it will distort the bacterial cells. 6  Staining Steps ◉ Cover the smear with methylene blue or safranin and allow the dye to remain in the smear for approximately one minute (Staining time is not critical here; somewhere between 30 seconds to 2 minutes should give you an acceptable stain, the longer you leave the dye in it, the darker will be the stain). ◉ Using distilled water wash bottle, gently wash off the excess dye from the slide by directing a gentle stream of water over the surface of the slide. ◉ Wash off any stain that got on the bottom of the slide as well. ◉ Saturate the smear again but this time with Iodine. Iodine will set the stain ◉ Wipe the back of the slide and blot the stained surface with bibulous paper or with a paper towel. ◉ Place the stained smear on the microscope stage smear side up and focus the smear using the 10X objective. ◉ Choose an area of the smear in which the cells are well spread in a monolayer. Center the area to be studied, apply immersion oil directly to the smear, and focus the smear under oil with the 100X objective. 7 Oil immersion lens 8  Thanks! ANY QUESTIONS? 9  ‫المعهد التكنولوجي‬ ‫العالي للعلوم الصحية‬ ‫التطبيقية ببني سويف‬ Gram Staining Prepared By: Amira Aly Supervisor: Dr. Manal Mohamed Abdelalim  Questions From the Previous Section 1- The simple stain can be used as a quick and easy way to determine the ………….... , ………….. , …………….. 2- Why we use a basic dye (Cationic dye) in the staining of bacteria? 3- Draw the arrangement of cocci, diplococci, tetrads and streptococci. 2  Gram Staining ◉ Gram Staining is the common, important, and most used differential staining techniques in microbiology. This test differentiate the bacteria into Gram Positive and Gram Negative Bacteria according to the chemical and physical properties of their cell wall, which helps in the classification and differentiations of microorganisms. 3 4  Principle of Gram staining When the bacteria is stained with primary In case of gram negative bacteria, cell stain Crystal Violet and fixed by the wall also takes up the CV-Iodine mordant, some of the bacteria are able to complex but due to the thin layer of retain the primary stain and some are peptidoglycan and thick outer layer decolorized by alcohol. The cell walls of gram which is formed of lipids, CV-Iodine positive bacteria have a thick layer of protein- complex gets washed off. When they sugar complexes called peptidoglycan and are exposed to alcohol, decolorizer lipid content is low. Decolorizing the cell dissolves the lipids in the cell walls, causes this thick cell wall to dehydrate and which allows the crystal violet-iodine shrink, which closes the pores in the cell wall complex to leach out of the cells. Then and prevents the stain from exiting the cell. when again stained with safranin, they So the ethanol cannot remove the Crystal take the stain and appears red in color. Violet-Iodine complex that is bound to the thick layer of peptidoglycan of gram positive bacteria and appears blue or purple in color. 5 ◉ During the crystal violet-iodine step, the bound molecules within the peptidoglycan of the gram + cell wall and within the membrane are held tightly. The acetone-alcohol actually causes the peptidoglycan molecules (arranged in a latticework) to shrink, thereby holding the crystal violet-iodine even tighter. In the gram negative cell, the outer lipopolysaccharide layer of the wall is dissolved by the decolorizer agents, and because the peptidoglycan layer is so thin in that group of bacteria, the crystal violet is leached out of the wall. 6 Summary of Gram Staining Steps 7  Reagents Used in Gram Staining ◉ Crystal Violet, the primary stain ◉ Iodine, the mordant ◉ A decolorizer made of acetone and alcohol (95%) ◉ Safranin, the counterstain 8  Preparation of Bacterial Smear 1. Draw a circle on clean and grease free microscopic slide and flip it over in order the circle to be on bottom 2. Add a drop of dist.H2O inside the circle of slide. 3. Using a sterilized inoculating loop to transfer an isolated colony from a culture plate to a slide with the water drop. 9  Preparation of Bacterial Smear 4. Disperse the bacteria on the loop in the drop of water on the slide and spread the drop over an area the size of a dime. It should be a thin, even smear. 5. Allow the smear to dry thoroughly until you see thin white haze. 6. Heat-fix the smear cautiously by passing the underside of the slide through the burner flame two or three times. It fixes the cell in the slide. Do not overheat the slide as it will distort the bacterial cells. 10  Staining Steps ◉ Cover the smear with Crystal Violet and allow the dye to remain in the smear for approximately 30 seconds to 1 minute. Wash with water ◉ Flood the gram’s iodine for 1 minute and wash with water. ◉ Then , Decolorize with 95% alcohol or acetone for about 10-20 seconds and rinse with water. ◉ Add safranin for about 1 minute and wash with water. ◉ Air dry, Blot dry and Observe under Microscope ◉ Place the stained smear on the microscope stage smear side up and focus the smear using the 10X objective. ◉ Choose an area of the smear in which the cells are well spread in a monolayer. Center the area to be studied, apply immersion oil directly to the smear, and focus the smear under oil with the 100X objective. 11 Oil immersion lens 12 13  Common Questions Q1. Give a few examples of gram-positive bacteria? Ans1. Gram-positive bacteria include the bacteria of genre Staphylococcus, Streptococcus, Enterococcus. Q2. Which is more harmful- gram-positive bacteria or gram-negative bacteria? Ans2. Gram-negative bacteria are more harmful and cause certain diseases. They are more intrinsically resistant to antibiotics - they don't absorb the toxin into their insides. Their ability to resist traditional antibiotics make them more dangerous in hospital settings, where patients are weaker and bacteria are stronger. Q3. Is it easier to kill gram-positive bacteria? Ans3. The cell wall of the gram-positive bacteria absorbs antibiotics and cleaning products. Because of the outer peptidoglycan layer, they are easier to kill. However Gram-negative bacteria cannot be killed easily. Q4. What infections are caused by gram-positive bacteria? Ans4. Gram-positive bacteria usually cause Urinary Tract Infections. These are caused commonly in people who are more prone to urinary tract infections or are elderly or pregnant. Q5. Which infections are caused by gram-negative bacteria? Ans5. The gram-negative bacteria cause various infections in humans such as indigestion, food poisoning, pneumonia, meningitis and other bacterial infections in the blood cells, bloodstream, wound infections, etc. The infections are caused by Acinetobacter, Pseudomonas aeruginosa and E.coli. 14  Errors!!! 1. Overheating (burning) during fixation can be avoided by carefully touching the back of the slide to the back of the hand each time the slide has been passed though the flame. 2. Do not stain smears which have only been air dried. Smears must also be "fixed". 3. Smears should not be too thick. After air drying, examine under a microscope. If there are no areas of bacteria separation, more water should be added to dilute the smear. 4. After staining it is essential that the back surface of the slide is wiped clean. 5. If washing with distilled water is not done adequately, crystallization of the stain may appear on the slide. 15 16 17 Colony Morphology 18  Thanks! ANY QUESTIONS? 19