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TECHNICAL INSTITUE FOR HEALTH TRAINING

(Practical Manual)

MIC 101

Prepared by: M.Homam.Jarkash (Microbiologist ) Supervised by : Dr.Sameh Rabia(Pharmacist )

2010

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Introduction

I hope this brief handout in practical pharmaceutical microbiology to be beneficial and will informative for pharmacy diploma in the health institutes.

It concerns about general aspects in pharmaceutical microbiology, for example: sterilization and disinfection, Gram staining and microscopy, general microbiological procedures, medical specimens collections and preparation of different important media.

I am asking Allah for conciliation to all of us.

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General safety

Some of the microorganisms used in this course may be pathogenic

for humans and animals. As a result, certain rules are necessary to avoid the possibility of infecting yourself or other people. A. Microbiological procedures:

1. reporting all spills and broken glassware to the instructor and receiving instructions for cleanup

2. methods for aseptic transfer 3. minimizing or containing the production of aerosols 4. washing hands prior to and following laboratories and at any

time contamination 5. never eating or drinking in the laboratory 6. disinfecting lab benches prior to and at the end of lab session 7. identification and proper disposal of different types of waste 8. never applying cosmetics, including contact lenses, or placing

objects (fingers, pencils) in the mouth or touching the face 9. good lab practice, including returning materials to proper

locations, proper care and handling of equipment.

B. Protective procedures: 1. tying long hair back, wearing personal protective equipment (eye

protection, coats, closed shoes; glasses may be preferred to contact lenses)

2. always using appropriate pipetting devices and understanding that mouth pipetting is forbidden

C. Emergency procedures: 1. locating and properly using emergency equipment (eye-wash

stations, first-aid kits, fire extinguishers, chemical safety showers, telephones, and emergency numbers)

2. reporting all injuries immediately to the instructor.

Universal Precautions and Laboratory Safety Precautions: 1- All specimens of blood and body fluids should be put in a well-

constructed container with a secure lid to prevent leaking during transport.

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2- All persons processing blood and body-fluid specimens should wear gloves.

3- For routine procedures, such as histologic and pathologic studies or microbiologic culturing, a biological safety cabinet is not necessary.

4- Mechanical pipetting devices should be used for manipulating all liquids in the laboratory.

5- Use of needles and syringes should be limited to situations in which there is no alternative.

6- Laboratory work surfaces should be decontaminated with an appropriate chemical germicide after a spill of blood or other body fluids and when work activities are completed.

7- Contaminated materials used in laboratory tests should be decontaminated before reprocessing or be placed in bags and disposed of in accordance with institutional policies for disposal of infective waste.

8- Scientific equipment that has been contaminated with blood or other body fluids should be decontaminated and cleaned before being repaired in the laboratory or transported to the manufacturer.

9. All persons should wash their hands after completing laboratory activities and should remove protective clothing before leaving the laboratory.

10. There should be no eating, drinking, or smoking in the work area.

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Session 1: Sterilization and Disinfection:

Sterilization: is the killing of all microorganisms in a material or on the

surface of an object - A surface or an object is either sterile or it is not sterile, there are no

gradations in sterility

Disinfection: means reducing the number of viable microorganisms present in a sample

- Not all disinfectants are capable of sterilizing, but, of course, all disinfectants are employed with the hope of disinfecting.

Sanitization: is the cleaning of pathogenic microorganisms from public eating utensils and objects such as that done by the kitchen of a restaurant.

Disinfectant: is a chemical or physical agent that is applied to inanimate objects to kill microbes.

Antiseptic: is a chemical agent that is applied to living tissue to kill microbes - Note that not all disinfectants are antiseptics because an antiseptic

additionally must not be so harsh that it damages living tissue. - Examples of Specific antimicrobial agents that used as

disinfectant or antiseptic: (i) Surfactants: soaps (ii) Various organic acids and bases: benzoic acid (iii) Heavy metals: mercury (mercurochrome) (iv) Halogen-containing compounds: Iodine, Chlorine (v) Alcohols: ethyl alcohol, propyl alcohol (vi) Phenol and phenol derivatives: Phenol, Dettol ® (vii) Oxidizing agents: Hydrogen peroxide (viii) Alkylating agents: formaldehyde, glutaraldehyde. (ix) Certain dyes: Gentian violet.

-Methods of Sterilization: 1- Heat:

Heat is a highly efficient means of sterilization so long as the material to be sterilized is resistant to heat.

- Different types of heat application include: A- Dry heat:

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To effect sterilization dry heat typically requires higher temperatures than moist heat It also is less penetrating and requires longer exposure.

-Typically one bakes materials in an oven at (i) 171C for at least one hour

(ii) 160C for at least two hours

(iii) 121C for at least 16 hours

Incineration is another common method of dry heat sterilization, e.g., such as the flame incineration of an inoculating loop.

Figure 1: Different types of dry heat ovens. B- Moist heat Moist heat is more effective than dry heat at a given temperature or

length of exposure Moist heat is also more penetrating than dry heat. However, to achieve sterilization employing moist heat requires rather elaborate equipment, i.e., the employment of an autoclave.

- Autoclave: is a high pressure device used to allow the application of moist heat

above the normal-atmosphere boiling point of water. Exposure to 121C for 15+ minutes is typically sufficient to sterilize, the material must be

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121C before the clock starts, Large items, large volumes, and items that are poorly penetrated by steam may take much longer than 15 minutes to sterilize.

Figure 2: Different types of autoclaves. C- Pasteurization:

is the application of moist heat of less-than boiling temperatures to foods to prevent the growth of food-spoiling organisms as well as various heat-labile pathogens.

2- Ultraviolet (UV) radiation: UV light is not terribly penetrating but is good for disinfecting surfaces and air.

3- Ionizing radiation Ionizing radiation is radiation that ionizes water; this temporarily turns water into an oxidizing agent.

4- Strong visible light Strong visible light can negatively affect bacterial viability so excessive exposure to strong visible light should be avoided when one's goal is culture preservation

5- Filtration (HEPA filters)

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Filtration is a common means of antimicrobial treatment used when materials are heat labile. High-efficiency particulate air (HEPA) filters are used to filter the air flowing into aseptic environments and out of potentially contaminated ones (e.g., containment facilities).

6- Gas Sterilization: It is a common means of antimicrobial treatment used when materials are heat and filtration labile. The most common used gas is: Ethylene oxide.

Effect of disinfectant –Soap- on Bacteria (Hand Washing):

Learning Objectives: 1. Understand the value of proper hand washing 2. Understand the importance of efficient decontamination procedures 3. Evaluate the effectiveness of hand washing and decontamination 4. Perform routine microbiological monitoring. Principles: Most commercial hand washing products contain antibacterial chemicals. Proper hand-washing technique performed by clinical personnel is the most effective method of controlling infections, especially nosocomial infections. A layer of oil and the structure of the skin prevent the removal of microorganisms by simple hand washing. Using a soap or gel will help remove the oil, and scrubbing with a brush for 7 to 8 minutes will maximize the removal of both transient(contaminated) and resident microorganisms. Procedure: 1st Period:

1- Before doing any hand washing, using one of your hands, gently make a five-finger impression on one of the TSA plates by rolling each finger and your thumb on the agar. Label this plate with your name, date, and “before”.

2- Take one of the soaps supplied and wash your hands according to the directions of your instructor. Wash your hands for the length of time assigned by the instructor. Some appropriate intervals are 10 seconds, 30 seconds, and 3 minutes.

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3- After washing, make another five-finger impression (using the same hand and fingers) on a different TSA plate. Mark this plate with your name, date, and the duration of hand washing and label it "after".

4- Incubate both plates at 35°C for 24 to 48 hours.

2nd Period:

Examine the TSA plates around the area where the finger

impressions were made. Compare the “before” and “after” plates. Results: a. Type of hand-washing material used: _____________________________________________________ b. Length of time of hand washing: ________________________________________________________ c. Type of culture medium used: __________________________________________________________ d. Hours of incubation: __________________________________________________________________ e. Temperature of incubation: _____________________________________________________________ f. Colony count before to hand washing: _____________________________________________________ g. Colony count after hand washing: _______________________________________________________ h. Colony shape : _________________________________________________________ I. Interpretation:

Session 2:

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Microscopy and bacterial staining

The microscope The microscope is a devise that magnifies the small unseen objects. So in order to study microbes we have to use microscopes. Types of microscopes:

1- light microscopes: 2 types 2- electron microscopes(EM): 2 types

The light microscope The light microscope uses the light and lenses to illustrate and magnified the objects. The simple light microscope consists of one lens that used to magnify the objects. The compound light microscope uses two systems of lenses. The typical objectives have powers of 10X, 40X, and100X.

Figure1.1: the compound microscope

In the compound microscope light from the source of light (lamp) is collected by condenser lens and pass to the specimen. The light pass the specimen to the objective lens that magnifies the image and passes it to the ocular lens that magnifies the image again and passes it to the eye. So the magnification power of a microscope is obtained by multiplying the magnification power of objective lens by the magnification power of eye lens. The typical ocular lens has magnification power of 10X.

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Electron microscope The EM was firstly made in Germany (1930-1933); but it began to be used for the studying of cells and tissue at 1952.The EM uses the electron beam instead of light.

Microscopic measurement of organisms:

Applications of light microscope 1- Wet mount preparation: in this process a drop of the microbial

suspension is placed on slide and covered by coverslip then it will examined by the light microscope.

2- Hanging-drop: a drop of the microbial suspension is placed on the coverslip, which is then inverted over the dip in a depression slide. Then it will be examined by the light microscope. The two

It frequently is necessary to accurately measure the size of the microorganism one is viewing. The size of microorganisms is generally expressed in metric units and is determined by the use of a microscope equipped with an ocular micrometer. An ocular micrometer is a small glass disk on which uniformly spaced lines of unknown distance, ranging from 0 to 100, are etched. The ocular micrometer is inserted into the ocular of the microscope and then calibrated against a stage micrometer, which has uniformly spaced lines of known distance etched on it. The stage micrometer is usually divided into 0.01 millimeter and 0.1 millimeter graduations. The ocular micrometer is calibrated using the stage micrometer by aligning the images at the left edge of the scales.

The dimensions of microorganisms in dried, fixed, or stained smears tend to be reduced as much as10 to 20% from the dimensions of the living microorganisms. Consequently, if the actual dimensions of a microorganism are required, measurements should be made in a wet-mount.

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Review questions Procedures that are needed to see microorganism by light microscope are:

Draw a representative field for each bacterium. E.coli

Streptococcus.epidermis

Use Magnification: X10 then Magnification: X40and X100

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Session 3: STAINING BACTERIA

The bacterium cell is very small it range from 0.1µm to 10 mm but there are some large bacteria that can be as long as 100µm.In order to study the size and shape of bacteria the bacteria fixed on a slide then it must be stained to get a contrast between the bacteria and its surrounding medium.

Simple stain

Bacteria can be stained using basic dyes like crystal violet, carbolfuchsin, and methylene blue. Simple dyes used for staining the whole cell or a part of it: example the spores can be stained by malachite green or carbolfuchsin. Crystal violet and others stain the 3whole cell.

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Differential stains

Use to distinguish between two or more cell types. Gram stain and acid-fast stain are other deferential stains Gram stain

The cell wall is stained in this process. Bacteria are stain as follows

1- stain with basic dye 2- iodine is added as a mordant for the stain 3- rinse with alcohol 4- stain with counter stain of deferent color of the initial stain.

If the bacteria appear with color on initial stain it said to be gram-positive, and if appear with color of the counter stain it said to be gram-negative bacteria. This is depends on the amount of peptidoglycan in the cell wall. gram-positive bacteria have large amount of peptidoglycan so it will absorbs the initial stain and alcohol can rinse it. Gram-negative bacteria have small amount of peptidoglycan so it will absorbs small amount of the initial stain so alcohol rinse it and it will absorbs the counter stain and retained its color.

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Acid-fast stain

It used in the identification of mycobacterium species. Mycobacterium is stained as follow:

1- stained with hot carbolfuchsin (red dye) 2- washed with acidified alcohol 3- stained with methylene blue

Acid-fast bacteria contain a wax-like material that binds a primary dye such as carbolfuchsin, even when they washed by acidified alcohol. So it will appear with red color. Nonacid- fast bacteria initially stained with the red dye but are decolorized by alcohol then it will stain with methylene blue. So it will appear with blue color.

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Review questions

1.What is the difference between a simple and differential stain?

- 2. Name the reagent used and state the purpose of each of the following in

the Gram stain: a. mordant b. primary stain c. decolorizer d. counterstain

3. Which step is the most crucial or most likely to cause poor results in the Gram stain? Why?

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4. Why must young cultures be used when doing a Gram stain?

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5.What is the purpose of the heat during the acid-fast staining procedure?

6. What is the function of the counterstain in the acid-fast staining procedure?

Make a drawing of the distribution of the colonies

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Session 4: Growth medium

A growth medium or culture medium is a liquid or gel designed to support the growth of microorganisms or cells [1], or small plantslike the moss Physcomitrella patens [2]. There are different types of media for growing different types of cells.

There are two major types of growth media: those used for cell culture, which use specific cell types derived from plants or animals, andmicrobiological culture, which are used for growing microorganisms, such as bacteria or yeast. The most common growth media for microorganisms are nutrient broths and agar plates; specialized media are sometimes required for microorganism and cell culture growth.[1] Some organisms, termed fastidious organisms, require specialized environments due to complex nutritional requirements. Viruses, for example, are obligate intracellular parasites and require a growth medium containing living cells.

Types of growth media The most common growth media for microorganisms are nutrient broths (liquid nutrient medium) or LB medium (Lysogeny Broth). Liquid media are often mixed with agar and poured intopetri dishes to solidify. These agar plates provide a solid medium on which microbes may be cultured. They remain solid, as very few bacteria are able to decompose agar. Bacteria grown in liquid cultures often form colloidal suspensions.

A. Minimal media

Minimal media are those that contain the minimum nutrients possible for colony growth, generally without the presence of amino acids, and

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are often used by microbiologists and geneticists to grow "wild type" microorganisms. Minimal media can also be used to select for or againstrecombinants or exconjugants.

Minimal medium typically contains:

a carbon source for bacterial growth, which may be a sugar such

as glucose, or a less energy-rich source like succinate

various salts, which may vary among bacteria species and growing conditions; these generally provide essential elements such as magnesium, nitrogen, phosphorus, and sulfur to allow the bacteria to synthesize protein and nucleic acid

water B. Selective media

Selective media are used for the growth of only select microorganisms. For example, if a microorganism is resistant to a certainantibiotic, such as ampicillin or tetracycline, then that antibiotic can be added to the medium in order to prevent other cells, which do not possess the resistance, from growing. Media lacking an amino acid such as proline in conjunction with E. coli unable to synthesize it were commonly used by geneticists before the emergence of genomics to map bacterial chromosomes.

Selective growth media are also used in cell culture to ensure the survival or proliferation of cells with certain properties, such as antibiotic resistance or the ability to synthesize a certain metabolite. Normally, the presence of a specific gene or an allele of a gene confers upon the cell the ability to grow in the selective medium. In such cases, the gene is termed a marker.

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Some examples of selective media include:

Eosin-methylene blue agar (EMB) that contains methylene blue – toxic to Gram-positive bacteria, allowing only the growth of Gram negative bacteria

YM (yeast and mold) which has a low pH, deterring bacterial growth

Blood agar (used in strep tests), which contains bovine heart blood that becomes transparent in the presence of hemolyticStreptococcus

MacConkey agar for Gram-negative bacteria

Hektoen enteric agar (HE) which is selective for Gram-negative bacteria

Mannitol salt agar (MSA) which is selective for Gram-positive bacteria and differential for mannitol

Terrific Broth (TB) is used with glycerol in cultivating recombinant strains of Escherichia coli.

Xylose lysine desoxyscholate (XLD), which is selective for Gram-negative bacteria

Buffered charcoal yeast extract agar, which is selective for certain gram-negative bacteria, especially Legionella pneumophila

C. Differential media

Differential media or indicator media distinguish one microorganism type from another growing on the same media.[5] This type of media uses the biochemical characteristics of a microorganism growing in the presence of specific nutrients or indicators (such as neutral red, phenol red, eosin y, or methylene blue) added to the medium to visibly indicate

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the defining characteristics of a microorganism. This type of media is used for the detection of microorganisms and by molecular biologists to detect recombinant strains of bacteria.

Examples of differential media include:

Eosin methylene blue (EMB), which is differential for lactose

and sucrose fermentation

MacConkey (MCK), which is differential for lactose fermentation

mannitol salt agar (MSA), which is differential for mannitol fermentation

X-gal plates, which are differential for lac operon mutants

Transport media

D. Enriched media

Enriched media contain the nutrients required to support the growth of a wide variety of organisms, including some of the more fastidious ones. They are commonly used to harvest as many different types of microbes as are present in the specimen. Blood agar is an enriched medium in which nutritionally rich whole blood supplements the basic nutrients. Chocolate agar is enriched with heat-treated blood (40-45°C), which turns brown and gives the medium the color for which it is named.

Media Preparation

1. Measure out approximately 90% of the final required volume of tissue culture grade water (Product No.W 3500), e.g. 900 ml for a final volume of 1000 ml. Select a container twice the size of the final volume.

2. While stirring the water add the powdered medium and stir until completely dissolved. Heating may be required to bring powders into solution.

3. Rinse the original container with a small volume of tissue culture grade water to remove traces of the powder. Add to the solution in Step 2.

4. Add desired heat stable supplements (e.g. sucrose, gelling agent, vitamins, auxins, cytokinins, etc.)

5. Add additional tissue culture grade water to bring the medium to the final volume.

6. While stirring, adjust medium to desired pH using NaOH, HCl or KOH.

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7. If a gelling agent is used, heat until the solution is clear. 8. Dispense the medium into the culture vessels before (or after) autoclaving

according to your application. Add heat labile constituents after autoclaving.

9. Sterilize the medium in a validated autoclave at 1 kg/cm2 (15 psi), 121 °C, for the time period described under Sterilization of Media Protocol.

10. Allow medium to cool prior to use.

Review questions List the steps you would go through to make tryptic soy agar slants. a. ________________________________________________________ b. _________________________________________________________ c. _________________________________________________________ d. _________________________________________________________ e. _________________________________________________________ f. _________________________________________________________

What are the three main types (in terms of their physical forms) of microbiological culture media? ____________________ ________________ ____________________

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Session5:

Section 2.1:- introduction

Microbial growth is the orderly increase in cellular constituents that result in the formation of new cell. This process depends on physiological capabilities of the species, the availability of nutrients, and the environmental conditions such as pH, temperature, and osmotic pressure. So to cultivate the microbes all the previous conditions must be available.

Section 2.2: - Pure culture A pure culture contains a single strain of microbe in which all cells are derived from a single parent cell. In nature microbes exist in mixed populations. For most purposes microbiologist must work with pure culture to ensure that the phenomena they observe are attributable to a given species. The techniques for growing pure culture depend on physically separating microbes in an environment that enable them to grow. This technique was discovered by Koch.

Section 2.3: - Isolation techniques A bacterium will grow into a distinct colony when it physically isolated on suitable solid growth medium. Agar is used as a solidifying agent for these media 1- The streak plate technique: It is common for isolating pure colonies of bacteria and yeast by streaking a sterile inoculating loop back and forth across the plate until the organisms are physically separated on the agar surface. The organisms grow into well-isolated

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colonies.

2- Pour plate

Pour plate can be made by repeated dilution of a bacterial culture in a tubes of melted agar at about 47 to 48 C. The content of each tube is poured into a petri dish and allowed to solidify.

Section 2.4: -Stock culture Pure culture of microorganisms can be maintained in stock culture for periods varying from weeks to years. Stock culture of bacteria, fungi, and algae are maintained on slant (used for aerobes) or in stabs (used for anaerobes) (figure 2.2). Stock culture prepared for long-term storage are either freeze-dried or stored in liquid nitrogen at –196 C or at –75 C in low temperature freezer.

Figure 2.2: agar slant and agar stab

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- Review What is the purpose of the spread-plate technique?

Make a drawing of the distribution of the colonies on each petri plate.

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Session 6: Collection of clinical specimens

Stool Specimen Collection Definition Stool specimen collection is the process of obtaining a sample of a patient's feces for diagnosic purposes. Purpose This procedure is used to test for infectious organisms, mucus, fat, parasites, or blood in the stool. Precautions Depending on the proposed analysis of the feces, watery feces will not be suitable for conducting a test for any fat that may be present, but can be used for other analyses, such as testing for bacteria. Description A stool specimen or culture can also be called a fecal specimen or culture. A specimen of freshly passed feces of 1/2 to 1 ounce (15 g to 30 g) is collected, without contamination of urine or toilet tissue, into a small container that may have a small spoon or spatula attached inside the lid of the cup for easier collection of the sample. Adult and older children patient can collect the specimen by passing feces into plastic wrap stretched loosely over the toilet bowl. A portion of the sample is then transferred into the supplied container. With young children and infants wearing diapers, the diaper should be lined with plastic wrap. A urine bag can be attached to the child to ensure that the stool specimen is not contaminated with urine. For a bedridden patient, the specimen should be collected in a bedpan lined with plastic wrap, and the nurse can transfer a portion of the feces into the appropriate container. Follow the manufacturer's guidelines if a commercial collection kit is used.

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Preparation If occult blood

is suspected, the patient should be given a

mild laxative

and should avoid eating foods rich in meat extracts

or leafy vegetables three days prior to the test. If the patient's gums bleed when brushing their teeth, the mouth should be cleansed with mouthwash and wiped with a cloth to avoid blood entering the digestive system and contaminating the stool specimen. Certain drugs may interfere with the analysis of the specimen, and the patient should avoid ingesting products such as antacids, oily foods and drugs, and antibiotics. Barium sulfate

should be excluded two weeks prior to the test, and medical procedure dyes three weeks prior to the test. If fat in the stool is suspected, the patient will also be asked to collect the samples in pre-weighed airtight containers. All feces passed in a 24-hour period are collected over two or three days and sent daily for analysis.

Urine Specimen Collection Definition The urine specimen collection

is a procedure used to obtain a sample of urine from a patient for diagnostic tests. Purpose The purpose of obtaining a urine sample is to test for any abnormalities that may be present, such as bacteria, ketones, or drugs. Precautions The skin of the genital area should be cleansed with a mild disinfectant to prevent contamination of the urine specimen or irritation of the delicate membranes of the area. Description A urine specimen is sometimes called a clean-catch, urine culture, or midstream specimen of urine, and is a method of collecting a quantity of urine for testing. Preparation

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The procedure and the reasons for it are explained to the patient. Able patients may be allowed to collect the urine sample, following the guidelines explained by the nurse. Nurses who collect the urine sample should be sure to wash and dry their hands carefully. The items required for the procedure are as follows:

a sterile urine cup for children and adults

a sterile urine bag for infants

a bedpan or urinal for patients unable to use the toilet

sterile swabs

sterile towels

sterile gloves For females, the area around the vulva is wiped and dried thoroughly with the sterile swabs and towels, working from front to back, with the nurse wearing sterile gloves. If the patient is unable to use the toilet, the bedpan is placed beneath her. When the urine begins to flow, the first part is allowed to pass into the toilet or bedpan. Then the sterile container is placed in position and filled with the mid-stream portion of the urine. The remainder of the urine is then allowed to pass into the toilet or bedpan. The lid is placed securely on the cup. For males, the area around the penis and urethra is wiped and dried thoroughly with the sterile swabs and towels, working from front to back, with the nurse wearing sterile gloves. If the patient is uncircumcised, the foreskin should be held back during the complete procedure to prevent the skin contaminating the sample. The patient then begins to pass urine into the toilet or a urinal. Then, the sterile container is placed in position and filled with the mid-stream portion of the urine, taking care that the penis does not touch the sides of the container. The remainder of the urine is then allowed to pass into the toilet or urinal. The lid is placed securely on the cup. For infants, the genitals are cleansed and dried thoroughly using the sterile wipes and towels. A sterile urine collection bag is placed over the area, with the adhesive tape firmly stuck onto the baby's skin. A fresh diaper is put on the child over the collecting bag and checked frequently for the child having passed urine into

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the bag. When the specimen is obtained, it is poured into a sterile container and sent immediately for testing.

Skin Specimen Collection Scrapings and Swabs In patients with suspected tinea or ringworm any ointments or other local applications present should first be removed with an alcohol wipe. Using a blunt scalpel, tweezers, or a bone curette, firmly scrape the lesion, particularly at the advancing border. A bone curette is safe and useful for collecting specimens from babies, young children and awkward sites such as interdigital spaces. If multiple lesions are present choose the most recent for scrapings as old loose scale is often not satisfactory. Any small vellus hairs when present within the lesions should be epilated. The tops of any fresh vesicles should be removed as the fungus is often plentiful in the roof of the vesicle. In patients with suspected candidiasis the young "satellite" lesions which have not undergone exfoliation are more likely to yield positive results if they are present. Otherwise the advancing scaly border should be scraped. When lesions in the flexures are moist and very inflamed it is more satisfactory and less painful to roll a moistened swab firmly over the surface. In patients with suspected cutaneous manifestations of systemic pathogens scrap the lesions with a bone curette or blunt scalpel as for tinea. A biopsy may be required in some cases. A. Skin scrapings: 1. Make a wet mount preparation in KOH for direct microscopy. Note a Calcofluor stained mount may also be necessary. 2. Inoculate specimens onto Sabouraud's dextrose agar slopes containing chloramphenicol and gentamicin, but NO cycloheximide and incubate at 35C. Maintain cultures for 4 weeks. B. Skin swabs: 1. Smear swab onto heat sterilized glass slide for Gram stain. 2. Inoculate specimens onto Sabouraud's dextrose agar containing chloramphenicol and gentamicin, but NO

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cycloheximide and incubate at 35C. Maintain cultures for 4 weeks. 3. Where secondary bacterial infection is suspected, and separate swabs for routine bacteriology were not collected, the swab should first be inoculated onto a blood agar plate, followed by the Sabouraud's agar containing the antibiotics and then placed into Brain Heart Infusion Broth. All cultures should be incubated at 35C. Maintain cultures for 4 weeks. Sputum Specimen Collection Definition Sputum specimen collection is a procedure designed to collect expectorated secretions from a patient's respiratory tract. Purpose Sputum is collected to be used as a laboratory specimen for the isolation of organisms that might be causing abnormalities of the respiratory tract. Precautions This procedure should not be performed if the patient is unable to take several deep breaths or cough deeply from the lungs. Description When secretions from the respiratory tract are expectorated, the secretions are called sputum. A sputum culture

is a sample of expectorated sputum. Induced sputum is a procedure to assist patients who have difficulty expectorating sputum. The patient inhales nebulized saline to loosen the sputum. To collect an induced sputum sample, the patient's mouth should be rinsed thoroughly with water

to reduce the amount of oral bacteria that are normally present from contaminating the sputum. The patient then inhales 20–30 ml of hypertonic saline from an ultrasonic nebuliser. The sputum is loosened and collected in a sterile sputum container. The patient should be supervised during the collection of the sputum to ensure the expectorated product has come from the lungs rather than saliva from the oral cavity. The sample is best taken first thing in the morning when the production of sputum is greatest.

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To collect an expectorated sputum sample, the patient should gargle and rinse out the mouth with water to reduce the amount of oral bacteria that are normally present from contaminating the sputum. The patient must take a deep breath and cough into a sterile sputum container. For a suspected common bacteria, one sputum sample may be required. If the suspected infection

is more complex, a sputum

sample may be required on three to five successive mornings. Preparation If there is any difficulty in expectorating, the physician may suggest the use of an inhalation, an expectorant, or physiotherapy

to aid in producing sputum for collection. The sputum should be transferred to the laboratory within two hours for analysis.

Results Sputum is mucoidal in appearance, resembling the thick liquid secreted by the mucous glands. It can be clear, white, or greenish in color, even blood stained. Blood in the sputum is called haemoptysis and may be a pink froth, mucus with a streak of blood, or an obvious clot, red in color representing fresh blood or brownish representing old blood. Haemoptysis may indicate that there has been some trauma to the respiratory tract, or that there is an infection present such as tuberculosis or even carcinoma. If it is determined that the blood is not from a simple cut to the mouth or a nosebleed, it is considered a serious condition and should be treated immediately. The sputum may also be frothy, indicating that the patient's pulmonary blood pressure is raised. Mucopurulent sputum contains mucus and pus and indicates an infection, such as an abscess, is present. There may be an unpleasant odor associated with sputum

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COLLECTION OF NASAL AND THROAT SWABS FOR RESPIRATORY VIRUS TESTING

SPECIMEN COLLECTION (the swabs will be combined for testing) • Label the VT or UTM tubes with the patient name, date of birth, collection date, specimen site (nose or throat) Nasal swab (collected from the nasal septum, not just the anterior nares) 1. Stand at the side of the patient 2. Ensure the patient’s head is resting against the wall 3. Place your hand on the patient’s forehead (non-dominant hand) and the thumb at the tip of the nose 4. Use a viral swab and insert the swab into the closest nostril horizontally, approximately 2–3 cm 5. Place sideways pressure on the swab in order to collect cells from the midline nasal septum 6. Rotate the swab twice (2 × 360° turns) collecting the epithelial cells (not mucous) 7. Place swab into the labelled VT or UTM tube (if UTM, fully insert the swab into the tube, snap the swab at the breakpoint, discard the residual shaft) and tighten the cap.. Throat swab 1. Stand at the side of the patient 1. Ensure the patient’s head is resting against the wall 2. Place your hand on the patient’s forehead (non-dominant hand) 3. Ask the patient to open their mouth widely and say ‘argh’ 4. Use a viral swab and insert the swab into mouth avoiding any saliva 5. Place sideways pressure on the swab in order to collect cells from the tonsillar fossa at

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the side of the pharynx 6. Rotate the swab twice (2 × 360° turns) collecting the epithelial cells (not mucous) 7. Place swab into the labelled VT or UTM tube (if UTM, fully insert the swab into the tube,

LABORATORY COLLECTION MANUAL EAR CULTURE I. GENERAL PRINCIPLE Culture of the external auditory canal usually reflects the microbial flora of the skin. The middle and inner ear should be sterile. There are several types of otitis externa. An acute localized infection in the form of a pustule or furuncle is due to Staphylococcus aureus. Erysipelas is caused by Group A strep and involves the external ear canal and soft tissue of the ear itself. Swimmer's ear is acute and diffuse and related to maceration of the ear from swimming or hot, humid weather. Gram negative bacilli, particularly Pseudomonas aeruginosa, are implicated. Chronic otitis externa is due to irritation from drainage of the middle ear in patients with chronic, suppurative otitis media and a perforated eardrum. Most cases of acute otitis media occur in children, with pneumococcus and Haemophilus influenzae the most common isolates. Mastoiditis is a complication of chronic otitis media and yields predominantly anaerobic flora. II. SPECIMEN COLLECTION A. External Ear 1. Cleanse external ear with mild germicide to reduce contaminating skin flora. 2. Any discharge in the external canal is a suitable specimen, for aerobic culture only. Sample discharge with an aerobic culturette, remove cap, place swab in sleeve and squeeze bottom of sleeve so that transport fluid from the sponge moistens the swab. B. Otitis Media

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1. Tympanocentesis is a needle aspirate performed by a physician using sterile equipment. The bacteriological findings from children with acute otitis media have been consistent enough that there is little justification for routinely performing tympanocentesis. The procedure is usually reserved for patients with recurrent otitis media or chronic persistent otitis; and considered in neonate and the elderly since their etiologies are unpredictable. 2. If the eardrum is ruptured, exudate should be collected by inserting a sterile swab through an auditory speculum. C. Mastoiditis An anaerobic culturette should be used to collect the specimen during surgery. III. TRANSPORT A. Transport aerobic and/or anaerobic culturettes to the lab ASAP. B. If the volume of specimen obtained by needle aspirate is small, the syringe, without needle, may be transported to the lab immediately and hand delivered to the microbiology staff. C. As always, transport specimens to the lab in a biohazard bag.

Routine Microbiological Specimens

Specimens Physical Ex.

Microscopic Exam

Culture media

Urine (MSU) / morning sample (better)

-Aspect -Color -Bloody

Wet film : - Pus cells - RBCs - Trichomones - Ova - Epithelial cells - Monilia - Nitrite

Gram's Stain :

Use standard loop of with sterile tip of automatic pipette (10 µl)

- MacConkey agar - Blood agar or CLED

Report colony count (CFU/ml): <102/ml not significant

>105/ml significant bacterium

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- if pus cells are numerous

102-105/ml doubtful significant

Throat mouth swab

Gram's stain :

- Pus cells - Candida - Borrelia vencenti

- Fusiform bacilli - Diphtheria

Routinely culture aerobic - Blood agar with

staphaureus streak or better incubate anaerobic.

- Add bacitracin dix

Sputum (Morning Sample)

- Blood streaks - purulent - mucous - Viscid - Salivary (rejected)

Wet film : - if salivary reject

sample (epith. cells > 10/LPF, Pus < 10/LPF)

Gram's stain : - ZN : if requested

- Blood agar (add optalim/anaerobe) - Chocolate agar - MacConkey

Wound swab or pus

Color Granules

Gram's stain: - Pus + bacteria - If gas gangrene

report at one

- Blood agar (aerobic) - MacConkey - Blood (anaerobic - on request for 5 days) - LJ on request

Vaginal swab cervical swab urethral discharge

Wet film : - For trichomonas, pus, RBCs and candida Gram's stain : - Vag. nugget score and clue cells - Urethral - PH - KOH test

Culture immediately:

- Prewarmed chocolate agar + CO2

- Blood agar (aerobic) & (anaerobic if request)

Prostatic secretion and semen

-Amount -Failed massage

Wet film Gram's stain

- Pre warmed chocolate + CO2

- Blood agar (aerobic)

Ear and

Gram's stain - Blood agar (aerobic)

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Eye swab

- pus + bacteria

- MacConkey - Blood agar

(anaerobic on request)

- Chocolate agar (ear) - Sabouraud agar (on

request)

Effusions

- Color - Aspect - Blood stained - Purulent - Chemical ex. (on request)

Cell number - Uncentrifuged - dil 1:1 --- no X 5 /mm3

wet film Gram's stain Leishman (type of cells) ZN

From sediment : - Chocolate agar +

CO2 - Blood agar (aerobic

& anaerobic / on request)

- MacConkey agar - LJ - Sabouraud on

request

CSF

- Color - Aspect - Blood stained - Chemical ex. (on request)

Cell count - mixed From deposit Wet film Gram's stain Leishman ZN *Keep CSF sample in incubator

- Pre warmed chocolate agar + CO2 3 days

- Blood agar - MacConkey agar - MacConkey agar

anaerobic (on request brain abces)

- Sabouraud agar on request

Stool

Wet film : - (pus, RBCs, Amoeba, cysts, larvae, eggs,…..)

Gram's stain - (campylobacter)

- XLD - MacConkey - Selenite Broth (18 h

in 37C) then subculture on MacConkey agar and XLD)

-Make a drawing of the crystals or parasites of each microscopic field

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References :

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Microbiology by Richard harve and Pamela champ LANGE by Jawetz, Melnick, & Adelberg Microbiology concepts and applications by Paul A ketchum Microbes in motion by Harry W seeley & john lee

Table of contents Introduction and General safety

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Session 1 Sterilization and Disinfection: 1) Heat 2) Moist heat 3) Autoclave: 4) Effect of disinfectant –Soap- on Bacteria (Hand Washing):

Session 2 1) Microscopy and bacterial staining 2) The light microscope 3) Microscopic measurement of organisms: 4) Applications of light microscope

Session 3: STAINING BACTERIA 1) Simple stain 2) Differential stains 3) Gram stain 4) Acid-fast stain

Session 4: Growth medium 1) Types of growth media 2) Minimal media 3) Selective media 4) Differential media 5) Enriched media 6) Media Preparation

Session5: Cultivation of bacteria 1) Pure culture 2) Isolation techniques 3) Pour plate 4) Stock culture

Session 6: Collection of clinical specimens 5) Stool Specimen Collection 6) Urine Specimen Collection 1) Skin Specimen Collection 2) Sputum Specimen Collection 3) COLLECTION OF NASAL AND THROAT SWABS FOR RESPIRATORY VIRUS TESTING

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