Chapter 4 Microbiological Laboratory Techniques Mosby items and

Microbiology Laboratory Microbes are everywhere in the environment. Necessary to ensure that organisms are selectively introduced into a culture and that other environmental organisms are not introduced. Ø Contaminant – a substance that is present in an environment where it does not belong or is present at levels that may cause harmful effects to living organisms. Mosby items and derived items © 2010 by Mosby, Inc. , an affiliate of Elsevier Inc. 2

Aseptic Technique Procedure performed under sterile conditions Prevents introduction of unwanted organisms or contaminants into an environment Ø Used when transferring microbes from one environment to another. Ø Prevents the contamination by fomites. Ø Fomite - any inanimate object or substance capable of transferring pathogens from one medium or individual to another. Mosby items and derived items © 2010 by Mosby, Inc. , an affiliate of Elsevier Inc. 3

Transferring Microbes Transfer loops and needles Ø Culture plates Ø Sterilized by flame or incinerator before and after use Hold in position that minimizes exposure of medium to the environment Test tubes Ø Removing lids/stoppers • Lid held in hand—flaming of lip of the test tube Mosby items and derived items © 2010 by Mosby, Inc. , an affiliate of Elsevier Inc. 4

Sterilization of Loops and Needles Mosby items and derived items © 2010 by Mosby,

Flaming of Test Tube Mosby items and derived items © 2010 by Mosby, Inc.

Sterilization Destruction or removal of all microorganisms Accomplished by Heat Ø Radiation Ø Filtration Ø Chemical methods Ø Sterile – items free of potentially contaminating microorganisms. Mosby items and derived items © 2010 by Mosby, Inc. , an affiliate of Elsevier Inc. 7

Sterilization - Heat Sterilization – Heat must reach a temperature of at least 121 degrees C. 20 -30 min. Ø 15 – 20 psi Ø Items must be heat resistant. Not suitable for moisture repellant items. Indicator tape is used to establish sterility Kills bacteria, viruses, fungi, and most fungal and bacterial fungal spores. Does not kill prions Mosby items and derived items © 2010 by Mosby, Inc. , an affiliate of Elsevier Inc. 8

Autoclave Mosby items and derived items © 2010 by Mosby, Inc. , an affiliate

Sterilization - Radiation Propagation of energy through space in the form of electromagnetic waves. Ø Ionizing radiation – disrupts the ionic and covalent bonding and denatures proteins(DNA). Gamma rays, electron beams. Ø Non-ionizing radiation – creates new covalent bonds which changes the structure of proteins (DNA) 10

Sterilization - Filtration is the mechanical separation of solids from fluid or gas by means of filters, which have various pore sizes. Used to sterilize sensitive material. Ø Vaccines, antibiotics, sera, cultured media. Fluids are pulled through a membrane to filters bacteria and viruses. Mosby items and derived items © 2010 by Mosby, Inc. , an affiliate of Elsevier Inc. 11

Sterilization - Chemical Low temperature gas sterilizers function by exposing the articles to be sterilized to high concentrations of very reactive gases (alkylating agents such as ethylene oxide). Liquid sterilants include bleach. Used on items that are sensitive to heat and moisture. Mosby items and derived items © 2010 by Mosby, Inc. , an affiliate of Elsevier Inc. 12

Disinfection Use of a physical process or chemical agent to destroy vegetative microbes and viruses Applied to: Inanimate surfaces Ø Medical equipment Ø Man-made objects. Ø Does not include endospores. • Bactericidal – substances that kill bacteria • Bacteriostatic - substances that interfere with cell growth Ø and reproduction. Antiseptics - are used to disinfect skin Mosby items and derived items © 2010 by Mosby, Inc. , an affiliate of Elsevier Inc. 13

Sanitization Any cleaning technique that mechanically removes microorganisms and other debris to reduce contamination to safe levels Sanitization uses compounds such as soap Ø Restaurants, dairies, breweries, and other food industries Mosby items and derived items © 2010 by Mosby, Inc. , an affiliate of Elsevier Inc. 14

Degermation Numbers of microbes on human skin are reduced by Scrubbing Ø Immersion in

The Five I’s Basic procedure to examine and characterize microbes Inoculation Ø Incubation Ø Isolation Ø Inspection Ø Identification Ø Mosby items and derived items © 2010 by Mosby, Inc. , an affiliate of Elsevier Inc. 16

The Five I’s Inoculation – The process of an inoculum being introduced to a culture. Ø Inoculum – a small sample of microorganisms. Mosby items and derived items © 2010 by Mosby, Inc. , an affiliate of Elsevier Inc. 17

The Five I’s Incubation – The placement of inoculated media in a proper environment.

The Five I’s Isolation – The process of separating a specific colony/ organism for

Mosby items and derived items © 2010 by Mosby, Inc. , an affiliate of

The Five I’s Inspection – observation and interpretation of morphology and growth patterns on different types of select media. Mosby items and derived items © 2010 by Mosby, Inc. , an affiliate of Elsevier Inc. 21

The Five I’s Identification – the process of specifying and recording traits of an

Media Provide nutrients for growth of organisms Some microbes require only simple inorganic compounds – non-fastidious Ø Others need a complex list of specific inorganic and organic compounds -fastidious At least 500 different types of media are used Mosby items and derived items © 2010 by Mosby, Inc. , an affiliate of Elsevier Inc. 26

Types of Media Physical state Liquid, semisolid, solid Ø Plate, broth, slant Ø Chemical state Complex or nonsynthetic Ø Chemically defined or synthetic Ø Functional type Ø General purpose, enriched, selective, differential Mosby items and derived items © 2010 by Mosby, Inc. , an affiliate of Elsevier Inc. 27

Liquid Media Water-based solutions that do not solidify at temperatures above freezing Tend to flow freely when container is tilted Growth occurs throughout the container These media are called broths Ø Growth is indicated by turbidity Mosby items and derived items © 2010 by Mosby, Inc. , an affiliate of Elsevier Inc. 28

Semisolid Media Are clot-like at room temperature They contain a small amount of solidifying agent—agar or gelatin Used to determine motility of bacteria Inoculated by stabbing the center of the medium with inoculating needle Mosby items and derived items © 2010 by Mosby, Inc. , an affiliate of Elsevier Inc. 29

Solid Media Dispensed in Petri plates Provide a firm surface for growth Discrete colonies Isolation and culturing bacteria and fungi Contains agar— polysaccharide isolated from red algae Ø Solid at room temperature Ø Liquefies at boiling temperature of water Mosby items and derived items © 2010 by Mosby, Inc. , an affiliate of Elsevier Inc. 30

Chemical Content of Media Chemically defined Termed synthetic Ø Contain pure organic and inorganic compounds Ø Chemical content specified by exact formula Ø Come in many forms Ø Standardized and reproducible Ø Mosby items and derived items © 2010 by Mosby, Inc. , an affiliate of Elsevier Inc. 31

Chemical Content of Media Complex media One or more components of a given medium is not chemically defined Ø Nonsynthetic Ø Cannot be represented by an exact chemical formula Ø Extracts from animals, plants, yeast, ground-up cells, tissues, and secretions—blood serum, meat extract, infusions Ø Mosby items and derived items © 2010 by Mosby, Inc. , an affiliate of Elsevier Inc. 32

Functional Types of Media General purpose media Grow broad spectrum of microbes that do not have special growth requirements Ø Nutrient agar and broth Ø Brain-heart infusion agar Ø Trypticase soy agar (TSA) Ø Mosby items and derived items © 2010 by Mosby, Inc. , an affiliate of Elsevier Inc. 33

Tryptic Soy Agar Complex medium used as an all-purpose growth medium Mosby items and

Functional Types of Media Enriched media Ø Contain complex organic substances, such as blood, serum, hemoglobin, or special growth factors Ø Blood agar – indicates hemolysis Ø Thayer-Martin agar – contains antibiotics and grows Neisseria sp. Ø Chocolate agar – Hemin and NAD which are found in blood cells. Mosby items and derived items © 2010 by Mosby, Inc. , an affiliate of Elsevier Inc. 35

Functional Types of Media Selective media Contain one or more agents that inhibit the growth of a certain microbe or microbes Ø Favor or select a certain microbe and allows it to grow Ø Isolation of a specific type of microorganisms from a complex sample such as feces, saliva, skin, water, and soil. Ø Mosby items and derived items © 2010 by Mosby, Inc. , an affiliate of Elsevier Inc. 37

Functional Types of Media Differential media Grow several types of microorganisms Ø Differences show up as variations in colony size or color, in media color change Ø Miscellaneous Reducing medium Ø Contain substances that absorb oxygen or slow the penetration of oxygen in a medium Ø Mosby items and derived items © 2010 by Mosby, Inc. , an affiliate of Elsevier Inc. 39

Xylose Lysine Deoxycholate Agar Chemically defined agar Both selective and differential Yellow Ø Carbohydrate xylose use by organism Black Ø Organism produces hydrogen sulfide Mosby items and derived items © 2010 by Mosby, Inc. , an affiliate of Elsevier Inc. 41

Inoculation Producing a culture Medium for inoculation is necessary Instrument used for inoculation must be sterile Inoculating loops or needles Sterile cotton swabs Mosby items and derived items © 2010 by Mosby, Inc. , an affiliate of Elsevier Inc. 42

Incubation Incubators with appropriate temperatures Most organisms grow at approximately 37° C Incubator temperature in the laboratory is generally between 20˚ C and 40˚ C Some organisms require higher temperatures —some require lower temperatures Mosby items and derived items © 2010 by Mosby, Inc. , an affiliate of Elsevier Inc. 43

Isolation Identification of bacteria requires isolating colonies Colonies that contain a single species of an organism—a pure culture Ø Can be obtained by three techniques: • Streak plate • Pour plate • Spread plate Mosby items and derived items © 2010 by Mosby, Inc. , an affiliate of Elsevier Inc. 44

Streak Plate Spreads millions of cells over the surface of a solid medium Separating individual cells Used for samples with a mixture of microorganisms Resulting isolated colonies can be used to produce pure cultures Mosby items and derived items © 2010 by Mosby, Inc. , an affiliate of Elsevier Inc. 45

Streak Plate Mosby items and derived items © 2010 by Mosby, Inc. , an

Pour Plate Used to produce isolated colonies Plate count—number of organisms in sample Inoculum is added to warm liquid agar in the Petri plate Plate allowed to cool and solidify Colonies will grow on surface and throughout the thickness of the agar Mosby items and derived items © 2010 by Mosby, Inc. , an affiliate of Elsevier Inc. 47

Pour Plate Mosby items and derived items © 2010 by Mosby, Inc. , an

Spread Plate Used to quantify the number of bacteria in a solution Small amount of inoculum is placed on agar plate and spread using a spreader stick Mosby items and derived items © 2010 by Mosby, Inc. , an affiliate of Elsevier Inc. 49

Spread Plate Spread plate after 24 hours of incubation Colonies are evenly distributed over the agar surface Colonies can easily be counted Mosby items and derived items © 2010 by Mosby, Inc. , an affiliate of Elsevier Inc. 50

Inspection The colonies or broth cultures are observed macroscopically for growth characteristics such as color, texture, and size Mosby items and derived items © 2010 by Mosby, Inc. , an affiliate of Elsevier Inc. 51

Identification Determination of the type of microbe Staining Specialized tests Biochemical Ø Immunological Ø Genetic Ø Mosby items and derived items © 2010 by Mosby, Inc. , an affiliate of Elsevier Inc. 52

Specimen Preparation Wet mounts Ø Live samples are placed on slides Ø Smear—a thin film of a solution of microbes on a slide Fixation Ø Heat fixation—to attach the microbes to the slide and to kill the microbes Staining Mosby items and derived items © 2010 by Mosby, Inc. , an affiliate of Elsevier Inc. 53

Negative Stain Negative stain Indirect staining process Ø Acidic stains—India ink or nigrosin Ø Dark background against organism Ø Stain does not require fixation of smear Ø Useful for staining • Spirochetes • Yeast capsules • Bacterial capsules Ø Mosby items and derived items © 2010 by Mosby, Inc. , an affiliate of Elsevier Inc. 54

Negative Stain: Klebsiella pneumoniae Mosby items and derived items © 2010 by Mosby, Inc.

Simple Stain Single stain applied to specimen after fixation onto the slide Most commonly used simple stains in microbiology include: Methylene blue Ø Crystal violet Ø Safranin Ø Mosby items and derived items © 2010 by Mosby, Inc. , an affiliate of Elsevier Inc. 56

Simple Stain (Crystal Violet): Bacillus cereus Mosby items and derived items © 2010 by

Differential Stains More complex than simple stains Use more than one stain to differentiate cellular components Used to demonstrate different staining characteristics of bacterial cells Allow differentiation between bacterial species Mosby items and derived items © 2010 by Mosby, Inc. , an affiliate of Elsevier Inc. 58

Gram Stain Most widely used differential stain in microbiology Most reliable with young bacterial cultures Four-step staining process Differentiates between gram-positive and gram-negative bacteria Gram-positive: purple Ø Gram-negative: red/pink Ø Mosby items and derived items © 2010 by Mosby, Inc. , an affiliate of Elsevier Inc. 59

Gram Stain Mosby items and derived items © 2010 by Mosby, Inc. , an

Acid-Fast Stained with carbolfuchsin and treated with acid-alcohol Acid-fast bacteria remain red Non—acid-fast—are blue when counterstained with methylene blue Mosby items and derived items © 2010 by Mosby, Inc. , an affiliate of Elsevier Inc. 61

Acid-Fast Stain Mosby items and derived items © 2010 by Mosby, Inc. , an

Special Stain Negative Ø Endospore Ø Used to demonstrate the presence of capsules Used to demonstrate the presence of endospores Flagella Ø Used to demonstrate the presence of flagella Mosby items and derived items © 2010 by Mosby, Inc. , an affiliate of Elsevier Inc. 63

Wright Stain Differential stain used for staining blood smears Mosby items and derived items

Identification Techniques Morphological characteristics Physiological characteristics Biochemical characteristics Mosby items and derived items ©

Morphology Used for preliminary identification of bacteria, fungi, parasites Morphological classification of bacteria Bacilli Ø Cocci Ø Spirilla Ø Vibrios Ø Spirochetes Ø Coccobacilli Ø Mosby items and derived items © 2010 by Mosby, Inc. , an affiliate of Elsevier Inc. 66

Culture Plates Nutrient agar Ø Tryptic (Trypticase) soy agar (TSA) Ø Commonly used general growth medium General purpose medium, base medium for other agar plates (i. e. , blood agar plates) Phenylethyl alcohol agar (PEA) Selective for gram-positive bacteria Ø Inhibits growth of gram-negative bacteria Ø Mosby items and derived items © 2010 by Mosby, Inc. , an affiliate of Elsevier Inc. 67

Culture Plates Blood agar plates (BAP) Ø Differentiate between species based on their ability to produce hemolysins Chocolate agar (CHOC) Blood agar plate Ø Used for growing difficult respiratory bacteria Ø Thayer-Martin agar (TM) Ø Chocolate agar for isolation of Neisseria gonorrhea and N. meningitides Mosby items and derived items © 2010 by Mosby, Inc. , an affiliate of Elsevier Inc. 68

Culture Plates Mac. Conkey agar (MAC) Ø Used to differentiate between gram-positive and gram-negative bacteria Xylose lysine deoxycholate (XLD) agar Culture of stool samples Ø Inhibits gram-positive bacteria—facilitates growth of gram-negative bacteria Ø Mannitol salt agar (MSA) Ø Differentiates organisms that ferment mannitol Mosby items and derived items © 2010 by Mosby, Inc. , an affiliate of Elsevier Inc. 69

Slants Culture tube with slanted medium surface After incubation amount of growth is None Ø Slight Ø Moderate Ø Abundant Ø Mosby items and derived items © 2010 by Mosby, Inc. , an affiliate of Elsevier Inc. 70

Growth Appearance on Slants Filiform Ø Echinulate Ø Uniform growth along the line of inoculation Margins of growth with jagged appearance Beaded Ø Separate or semiconfluent colonies appear along the line of inoculation Mosby items and derived items © 2010 by Mosby, Inc. , an affiliate of Elsevier Inc. 71

Growth Appearance on Slants Effuse Ø Arborescent Ø Thin, veil-like growth, usually spreading Branched, tree-like growth Rhizoid Ø Growth with root-like appearance Mosby items and derived items © 2010 by Mosby, Inc. , an affiliate of Elsevier Inc. 72

Colony Growth on Agar Mosby items and derived items © 2010 by Mosby, Inc.

Gelatin Stabs Use deep tubes containing 12% nutrient gelatin Heavy inoculum from pure culture is stabbed into the medium Incubated for at least 48 hours followed by 30 minutes of refrigeration If organism has produced gelatinase content, it will remain liquid or partially liquid Mosby items and derived items © 2010 by Mosby, Inc. , an affiliate of Elsevier Inc. 74

Producing a Gelatin Stab Mosby items and derived items © 2010 by Mosby, Inc.

Types of Liquefaction in a Gelatin Stab Crateriform Ø Saucer-shaped liquefaction Napiform Ø Infundibular Ø Funnel-like or inverted cone Saccate Ø Turnip-like Tubular or cylindrical elongated sac Stratiform Ø Liquefied to the walls of the tube in upper region Mosby items and derived items © 2010 by Mosby, Inc. , an affiliate of Elsevier Inc. 76

Growth Patterns in Broths Bacteria grown in broth exhibit the following patterns Ø Sediment at the bottom of the tube Ø Turbid growth throughout the tube Ø Pellicle—a thick growth at the top of the tube Mosby items and derived items © 2010 by Mosby, Inc. , an affiliate of Elsevier Inc. 77