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Microbiology Labs.

Microbiology Labs.

Bacteria in the laboratory.

Maleeha Fatima

August 27, 2023

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  1. 1 BACTERIA  Bacteria (common noun bacteria, singular bacterium) are

    a type of biological cell.  They constitute a large domain of prokaryotic microorganisms.  Bacteria are typically few micrometers in length.  The study of bacteria is known as bacteriology, a branch of microbiology. Shapes and arrangement of bacteria
  2. 2 GRAM STAINING It is common and easy method to

    identify the bacteria. The gram staining technique was developed by Danish bacteriologist Hans Christian Gram. The gram stain technique is based on the differential structure of the cellular membranes and cell walls of the two groups. The two major groups of bacteria can be divided into gram-positive and gram-negative. This technique utilizes four steps staining procedure with two different dyes to identify unknown species of bacteria. REAGENTS 1. Primary stain - Crystal violet 2. Mordant - Gram iodine 3. Gram decolorizer – Ethanol 4. Counter stain – Gram safranin
  3. 3 PROCEDURE Label a microscope slide Sterilize inoculation loop by

    flaming Add a drop of saline on the slide Flame the metal loop again to sterilize Transfer your test sample on the slide with syringe/cotton swab/wire loop Flame the metal loop again to sterilize Let the liquid completely evaporate Heat fix the bacteria to the slide by quickly passing the slide on the flame Add few drops of crystal violet on the smear and left the stain untouched for one minute
  4. 4 Rinse the slide with tap water Add few drops

    of gram iodine on the smear and left the stain untouched for one minute Rinse the slide with tap water Add few drops of decolorizer & let the decolorizer flow over the bacteria at a 45° angle until the flow clear Rinse the slide with tap water Add few drops of safranin on the smear and left the stain untouched for one minute Rinse the slide with tap water Allow the slide to dry it completely Observe the slide under a microscope by adding oil on the slide
  5. 5 RESULT Gram-positive organisms contain a highly cross-linked thick layer

    of peptidoglycan that retains the primary dye, crystal violet, following the application of the mordant, iodine. The iodine and crystal violet form a complex within the peptidoglycan. When decolorizer is applied to the cells, the Crystal violet-Iodine complex remains within the cell, making it appear dark purple to blue. The gram-negative organisms do not contain a thick cross-linked layer of peptidoglycan. The peptidoglycan is loosely distributed between the inner cell and the outer cell membranes. Following the application of the crystal violet and iodine, the Crystal violet-Iodine complexes are not trapped within the peptidoglycan. Application of the acid-alcohol decolorizer dehydrates the outer cellular membrane, leaving holes in the membrane and effectively washing or removing the CV-I complex from the cells. The cells appear colorless. To make the colorless cells visible, a secondary stain, safranin, is applied, leaving the gram-negative cells pink.
  6. 7 Microorganisms observed under microscope Gram –ve rods Gram +ve

    cocci E. coli. Klebsiella Pseudomonas Salmonella Proteus Enterobacter Streptococcus Staphylococcus
  7. 8 CULTURE MEDIA  The observable growth that appears in

    or on the medium is known as a culture.  The substances & environment, which is provided to the microorganisms for their growth is called as media.  Culture media are contained in test tubes, flasks, or Petri dishes.  Media are extremely varied in nutrient content & consistency & can be specially formulated for a particular purpose. TYPES OF MEDIA Media can be classified on the three primary levels: 1. Physically 2. Chemically 3. Functionally

    defined as water-based solutions that do not solidify at temperatures above freezing & that tends to flow freely when the container is tilted. These media termed broths, milks, or infusion, are made by dissolving various solutes in distilled water. Growth occurs throughout the container & can then present a dispersed cloudy or particulate appearance. Examples: Luria broth, Nutrient broth. 2- SEMISOLID MEDIA It exhibit clot-like consistency. It contain solidifying agent (agar or gelatin) that thickens the media but does not produce a firm substrate. This media is used to determine the motility of bacteria & to localize a reaction at a specific site. Examples: Both motility test medium & Sulfide Indole Motility (SIM) contain a small amount of agar. The medium is
  9. 10 stabbed carefully in the center & later observed for

    the pattern of growth around the stab line. 3- SOLID MEDIA It provides a firm surface on which cells can form discrete colonies & are advantages for isolating & sub- culturing bacteria & fungi. They come in two forms: Liquefiable, Non-liquefiable.  Liquefiable solid media (also known as reversible solid media) contains solidifying agent i.e. agar which is thermoplastic. Agar is solid at room temperature and it melts or liquefies at 100 °C.  Non-liquefiable solid media have less versatile applications than agar media because they are not thermoplastic. This type of media include materials such as rice grains (used to grow fungi), cooked meat media (good for anaerobes), potato slices etc. All of these media start out solid and remain solid after heat sterilization.

    compositions are chemically defined are termed as synthetic media. These media contain highly pure organic & inorganic compounds that vary little from one source to another & have a molecular content specified by means of an exact formula. Examples: Glucose-salt agar, Inorganic synthetic broth. 2- NON-SYNTHETIC MEDIA Media whose compositions are not chemically defined are termed as non-synthetic media. It contains at least one ingredient that is not chemically definable. Most of these substances are extracts of animals, plants, or yeasts including such materials as ground-up cells, tissues, secretions, milk, yeast extract, soybean digests, and peptone. Examples: Nutrient broth, Blood agar, EMB agar.

    are designed to grow as broad a spectrum of microbes as possible. They are nonsynthetic & contain a mixture of nutrients that could support the growth of pathogens & non pathogens alike. Examples: Nutrient agar, Nutrient broth, Brain-heart infusion, Trypticase soy agar. 2- ENRICHED MEDIA It contains complex organic substances (blood, serum, hemoglobin or special growth factors) that allow certain species to grow. Examples: Streptococcus pneumoniae is cultured on blood agar, which is made by adding sterile sheep, horse or rabbit blood to a sterile agar base, Neisseria can grow on Thayer-Martin medium or chocolate agar, which is essentially cooked blood agar.
  12. 13 3- SELECTIVE MEDIA It contains one or more agents

    that inhibit the growth of certain microbes thereby encourage the growth of desired microbe. This type of media is very important in primary isolation of a specific type of microorganism from samples containing a highly mixed population. Examples: Mannitol Salt Agar (MSA), MacConkey agar, Eosin-Methylene Blue (EMB) agar. 4- DIFFERENTIAL MEDIA A differential media can grow several types of microorganisms, but it is designed to highlight differences among these microorganisms. Its ability of differentiation is due to the type of agents added. Examples: Sulfide Indole Motility (SIM) agar, Xylose Lysine Deoxycholate (XLD) agar, Triple Sugar Iron (TSI) agar.
  13. 14 5- MISCELLANEOUS MEDIA  Reducing medium contains a substance

    (thioglycollic acid or cystine) that absorbs oxygen or slows the penetration of oxygen in a medium thus reducing its availability. These media are important for growing anaerobic bacteria or determining oxygen requirements. Example: Thioglycolate broth.  Carbohydrate fermentation media contain sugars that can be fermented (converted to acids) & a pH indicator to show this reaction.  Transport media is used to maintain & preserve specimens that have to be held for a period of time prior to clinical analysis or to sustain delicate species that die rapidly if not held under stable conditions. Example: Stuart’s & Amies transport media contain salts, buffers & absorbants to
  14. 15 prevent cell destruction by enzymes, pH changes & toxic

    substances but will not support growth.  Assay media are used by technologists to test the effectiveness of antimicrobial drugs & by drugs manufacturers to assess the effect of disinfectants, antiseptics, cosmetics & preservatives on the growth of microorganisms.  Enumeration media is used by industrial & environmental microbiologists to count the numbers of microorganisms in milk, water, food, soil & other samples.
  15. 16 Sample Media Urine MacConkey agar Cystine-lactose-electrolyte-deficient (CLED) agar Stool

    MacConkey agar Salmonella-Shigella agar Sputum Pus HVS Blood Tissue MacConkey agar Blood agar
  16. 17 MacConkey Agar Lactose fermenter colonies For Example: E. coli.

    Klebsiella Non-lactose fermenter colonies For Example: Pseudomonas Salmonella Proteus Shigella
  17. 21 Mannitol Salt Agar (MSA) plate Staphylococcus aureus on MSA

    ferments mannitol which causes phenol red to turn into yellow

    Urine S. No. Date Serial Number in Lab Register Microorganism Identified 1 1/9/2020 3 E. coli. 2 2/9/2020 9 Klebsiella 3 2/9/2020 10 E. coli. 4 3/9/2020 14 Klebsiella 5 3/9/2020 15 E. coli. 6 3/9/2020 16 Pseudomonas 7 3/9/2020 18 E. coli. 8 5/9/2020 25 Staphylococcus 9 5/9/2020 26 E. coli. 10 5/9/2020 28 E. coli.
  19. 23 11 5/9/2020 29 Pseudomonas 12 7/9/2020 31 E. coli.

    13 7/9/2020 37 E. coli. 14 7/9/2020 38 Klebsiella 15 7/9/2020 40 E. coli. 16 8/9/2020 47 E. coli. 17 8/9/2020 50 E. coli. 18 9/9/2020 58 Klebsiella 19 9/9/2020 61 Klebsiella 20 9/9/2020 62 E. coli. 21 10/9/2020 63 E. coli. 22 10/9/2020 67 E. coli. 23 10/9/2020 70 Pseudomonas 24 10/9/2020 72 E. coli. 25 10/9/2020 73 E. coli. 26 10/9/2020 74 Klebsiella
  20. 24 27 11/9/2020 83 E. coli. 28 12/9/2020 91 E.

    coli. 29 12/9/2020 92 E. coli. 30 14/9/2020 103 E. coli. 31 15/9/2020 105 E. coli. 32 15/9/2020 106 Pseudomonas 33 16/9/2020 109 Klebsiella 34 16/9/2020 113 E. coli. 35 16/9/2020 115 E. coli. 36 17/9/2020 119 E. coli. 37 18/9/2020 131 Pseudomonas 38 19/9/2020 135 E. coli. 39 19/9/2020 143 E. coli. 40 21/9/2020 157 Pseudomonas 41 22/9/2020 162 E. coli. 42 22/9/2020 164 E. coli.
  21. 25 43 23/9/2020 166 Pseudomonas 44 23/9/2020 167 E. coli.

    45 23/9/2020 179 Pseudomonas 46 25/9/2020 195 Pseudomonas 47 26/9/2020 199 E. coli. 48 26/9/2020 201 E. coli. 49 28/9/2020 212 E. coli. 50 28/9/2020 213 E. coli.
  22. 26 Sample: Pus S. No. Date Serial Number in Lab

    Register Microorganism Identified 1 1/9/2020 4 Pseudomonas 2 2/9/2020 8 Staphylococcus 3 2/9/2020 11 Staphylococcus 4 4/9/2020 21 Staphylococcus 5 7/9/2020 33 Staphylococcus 6 7/9/2020 35 Enterobacter sp. 7 7/9/2020 42 Staphylococcus 8 8/9/2020 43 Staphylococcus 9 8/9/2020 46 Staphylococcus 10 8/9/2020 49 Staphylococcus 11 9/9/2020 55 Staphylococcus 12 9/9/2020 56 Pseudomonas 13 10/9/2020 65 Pseudomonas Staphylococcus
  23. 27 14 10/9/2020 69 Staphylococcus 15 10/9/2020 71 Staphylococcus 16

    11/9/2020 82 Staphylococcus 17 14/9/2020 94 Proteus 18 14/9/2020 95 Pseudomonas 19 14/9/2020 96 Pseudomonas 20 14/9/2020 97 Staphylococcus 21 16/9/2020 111 Proteus 22 17/9/2020 121 Klebsiella 23 21/9/2020 149 Staphylococcus 24 21/9/2020 153 Staphylococcus 25 21/9/2020 156 Staphylococcus 26 23/9/2020 172 Pseudomonas 27 23/9/2020 173 Klebsiella 28 23/9/2020 177 Pseudomonas 29 23/9/2020 178 Klebsiella
  24. 28 30 25/9/2020 193 Staphylococcus 31 25/9/2020 196 Pseudomonas 32

    25/9/2020 197 Enterobacter sp. 33 26/9/2020 204 Enterobacter sp. 34 26/9/2020 205 Staphylococcus 35 28/9/2020 210 Staphylococcus Sample: Blood S. No. Date Serial Number in Lab Register Microorganism Identified 1 7/9/2020 21 Salmonella 2 9/9/2020 34 Klebsiella 3 9/9/2020 36 Salmonella 4 12/9/2020 47 Klebsiella 5 21/9/2020 83 Streptococcus
  25. 29 S. No. Date Sample Serial Number in Lab Register

    Microorganism Identified 1 1/9/2020 Tissue 7 E. coli. 2 11/9/2020 Sputum 89 Klebsiella 3 18/9/2020 HVS 132 Pseudomonas

    the release of oxygen from hydrogen peroxide. Catalase test is used to differentiate between bacteria that produce catalase from non-catalase producing bacteria. Using a sterile wire loop/cotton swab, the test organism was transferred in the test tube or the glass slide and a drop of 3% hydrogen peroxide onto the medium. Then observe the bubble formation. 2H2 O2 For example: Catalase +ve bacteria: Staphylococcus Catalase –ve bacteria: Streptococcus 2H2 O + O2 (gas)
  27. 32 OXIDASE TEST Oxidase test is used to determine if

    the bacterium produces cytochrome c oxidases. Disks/strip impregnated with a reagent TMPD or DMPD which is redox indicator. TMPD acts as an artificial electron donor for the oxidase. Cytochrome system is present in aerobic organisms that can use oxygen as the terminal electron acceptor. The end product of this metabolism is either H2 O or H2 O2. The test organism was picked with sterile wire loop/cotton swab and rubbed on the oxidase strip. As the oxidized reagent forms colored compound indophenol blue and if the test organism produce oxidase, the strip turns from white to purple or violet color within few seconds. For example: Oxidase +ve bacteria: Pseudomonas Oxidase -ve bacteria: E. coli. (does not have cytochrome c oxidase that’s why they not use oxygen for energy production).
  28. 33

  29. 34 INDOLE PRODUCTION TEST Indole is a nitrogenous compound formed

    as a result of bacterial breakdown of amino acid tryptophane. This breakdown requires the production of enzyme tryptophanase by certain bacteria. Indole production test is a qualitative procedure in which the test organism was inoculated in tryptone agar tube and was incubated at 37°C for 24 hours. After incubation, few drops of Kovac’s reagent then added in tube. When indole is combined with p-dimethyl aminobenzaldehyde in Kovac’s reagent and produces quinoidal, the top layer of the test tube turns from yellow to cherry red which means that the test organism has an ability to produce indole. Left side = indole –ve e.g. Klebsiella Right side = indole +ve e.g. E. coli.
  30. 35 CITRATE UTILIZATION TEST Simmon’s citrate agar slant was inoculated

    with test organism. The tube was incubated at 37°C for 24 hours. Bacteria that can grow on this medium use citrate and covert ammonium phosphate to ammonia and ammonium hydroxide, creating an alkaline pH. The pH change turns the bromothymol blue indicator from green to blue. Left side = citrate –ve e.g. E. coli. Right side = citrate +ve e.g. Klebsiella
  31. 36 UREASE TEST Urease test is useful diagnostic test for

    identifying bacteria, especially to distinguish members of the genus Proteus from gram negative pathogens. Proteus vulgaris is an important and fast producer of urease. Urease test is performed by growing test organism on urea agar slant with phenol red as indicator with pH 6.8. During the incubation period, the organism capable of producing urease enzyme hydrolyse urea and produce ammonia that raises the pH level. As the pH increases, the phenol red changes from yellowish to pinkish color. Left side = urease –ve e.g. E. coli. Right side = urease +ve e.g. Proteus
  32. 37 COAGULASE TEST Coagulase is an enzyme produced by Staphylococcus

    aureus that converts soluble fibrinogen in plasma to insoluble fibrin. Staphylococcus aureus produce two forms of coagulases, bound and free. Slide coagulase test is done to detect bound coagulase or clumping factor. Tube coagulase test is done to detect free coagulase. To perform coagulase test, a drop of plasma is added into a slide or test tube. Then test organism is transfer to that.
  33. 38 BILE SOLUBILITY TEST This test is a qualitative procedure

    for determining the ability of bacterial cells to lyse in the presence of bile salts. The test is primarily used to differentiate bile soluble Streptococcus pneumonia from bile insoluble alpha-hemolytic streptococci. In this test, the test organism is transfer into the tube containing bile. Positive result is indicated by the clear tube suspension, the negative result is indicated if the suspension remains turbid.
  34. 39 ZIEHL-NEELSEN STAIN This stain is used to identify Mycobacterium

    tuberculosis, which is an acid fast bacteria. These types of bacteria have a lipoid capsule that has a high molecular weight and is waxy at room temperature that makes the organism impenetrable by aqueous based staining solutions. The lipoid capsule of an acid fast organism stains with carbol-fuchsin and resists decolorization with dilute acid rinse. The acid fast bacilli will stain bright red. Reagents used in this procedure include:  Carbol-fuchsin  Acid alcohol  Methylene blue
  35. 40 ANTIBIOTIC SENSITIVITY TESTING Antibiotic susceptibility or sensitivity testing is

    the measurement of the susceptibility of bacteria to antibiotics as bacteria may have resistance to some antibiotics. Commonly for qualitative antibiotic sensitivity testing, the disc diffusion method is used. In this method, antibiotic- impregnated discs are placed on agar plate (Mueller-Hinton Agar frequently use for this purpose) having bacterial growth. This is called as the Kirby-Bauer method. If the antibiotic inhibits microbial growth, a clear zone called zone of inhibition is seen around the antibiotic disc but, if the bacteria will grow around antibiotics to which they are resistant, no zone of inhibition will be observed around antibiotic disc. MHA plates with bacterial growth and antibiotic discs Zone of inhibition around antibiotic disc Bacterial growth around antibiotic disc
  36. 41 COMMON ANTIBIOTICS USED IN LAB S. No. Antibiotic name

    Symbol Bacteria (gram stain) 1 Ampicillin AMP +/- 2 Amikacin AK +/- 3 Azteronam ATM +/- 4 Cefixime CFM +/- 5 Ceftazidime CAZ +/- 6 Ceftizoxime ZOX +/- 7 Ceftriaxone CRO +/- 8 Cefuroxime CXM +/- 9 Chloramphenicol C + 10 Ciprofloxacin CIP +/- 11 Clindamycin DA + 12 Cloxacillin OB + 13 CoTrimoxazole SXT +/-
  37. 42 14 Fosfomycin FOS +/- 15 Gentamycin CN +/- 16

    Imipenem IPM +/- 17 Levofloxcin LEV + 18 Linezolid LZD + 19 Meropenem MEM +/- 20 Nalidixic acid NA For urine only 21 Nitrofurantoin F For urine only 22 Norfloxacin NOR For urine only 23 Sulzone SCI +/- 24 Tigecycline TGC +/- 25 Tozabaclam TZP +/- 26 Urixcin UR For urine only 27 Vancomycin V +