Laboratory procedures employed in the identification of bacteria

6.Isolation of organism in pure culture 7.Bacterial colony morphology 8.Microscopic morphology and Staining reaction 4. Biochemical test 5. Serological procedure 6. Antibiotic sensitivity

Isolation of organism in Pure Culture

Pure culture (axenic culture) – Population of cells arising from a single cell - the approach used for the isolation of organism depends upon the source of clinical specimen Blood, spinal fluid and closed abscesses may yield almost pure bacterial culture specimen of sputum, stool, materials from the skin and body orifices usually contains mixture of organism - Spread plate, streak plate, and pour plate are

Laboratory Cultivation
Cultivation is the process of growing microorganisms

by taking bacteria from the infection site by some means of specimen collection and growing them in the artificial environment of the laboratory For the in vitro environment of the bacteria, required nutrients are supplied in a culture medium culture - organisms that grow and multiply in or on a culture media

Culture Medium - is a liquid or gel designed to support the growth of microorganisms - 2 major types of growth media: - those used for cell culture, which use specific cell types derived from plants or animals - microbiological 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

Based on Chemical Composition Complex Media - Contain some ingredients of unknown composition and/or conc. - is a medium that contains: • carbon source such as glucose for bacterial growth • water • various salts needed for bacterial growth • a source of amino acids and nitrogen (e.g., beef, yeast extract) - Nutrient media contain all the elements that most bacteria need for growth and are non-selective, so they are used for the general cultivation and maintenance of bacteria kept in laboratory culture collections Defined or Synthetic Media -All components and their concentrations are known

Functional Types of Media Supportive or general purpose media
- Support the growth of many microorganisms - E.g., Tryptic soy agar

Enriched media - General purpose media supplemented by blood or other special nutrients

• 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

Selective media
- Favor the growth of only selected microorganisms and inhibit growth of others
• eosin-methylene blue agar (EMB) that contains methylene blue – toxic to Gram (+) bacteria, allowing only the growth of Gram (-) bacteria • blood agar (used in strep tests), which contains beef heart blood that becomes transparent in the presence of hemolytic Streptococcus • MacConkey agar for Gram-negative bacteria

Alpha Hemolytic Streptococci

Gamma Hemolytic Streptococci Beta Hemolytic Streptococci

Incomplete lysis of RBC’s

Complete lysis of RBC’s

No lysis of RBC’s

Differential media – Distinguish between different groups of microorganisms based on their biochemical characteristics 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 the defining characteristics of a microorganism Ex. • Blood agar – differentiates hemolytic versus non-hemolytic
bacteria • MacConkey agar - lactose fermenters versus non-fermenters • Eosin methylene blue (EMB), which is differential for lactose and sucrose fermentation • Mannitol Salt Agar (MSA), which is differential for mannitol fermentation

Bacterial colony morphology
• Bacteria  grow on solid media as colonies
• colony is defined as a visible mass of microorganisms all originating from a single mother cell, when inoculated into appropriate medium containing 2% agar and incubated 18-24 hours in a favorable atmosphere • therefore a colony constitutes a clone of bacteria all genetically alike • Ideally, the colony is the progeny of one, or at most, a few bacteria • A colony will usually contain millions of bacterial cells • Colony morphology can sometimes be useful in bacterial identification • Colonies are described as to such properties as size, shape, texture, elevation, pigmentation, effect on growth medium

To identify the following colonial characteristics/culture characteristics: WHOLE SHAPE OF COLONY EDGE/MARGIN OF COLONY

ELEVATION OF COLONY (turn the place on end to determine height)

CHROMOGENESIS (pigmentation)
- Some bacterial species form an array of pigments: white, red, purple, etc. • Some pigments are contained within the cell (i.e., probably not water soluble) • Some pigments readily diffuse throughout the medium (i.e, water soluble) • Some pigments fluoresce in UV light

transparent (clear), opaque, translucent (almost clear, but distorted vision–like looking through frosted glass iridescent (changing colors in reflected light)

butyrous (buttery), viscid (sticks to loop, hard to get off) brittle/friable (dry, breaks apart)

Is it easy or difficult to emulsify?  Does it form a uniform suspension, a

SURFACE OF COLONY: smooth, mucoid/glistening, rough, dull (opposite of glistening), rugose (wrinkled)

Smooth - colonies gives the appearance of homogeneity and
uniform texture without appearing as liquid or as mucoid colonies characteristically isolated from fresh wild type organism such as gram- negative enterobacteria Ex. Salmonella, Shigella

Mucoid - colonies exhibits a water-like glistening confluent
appearance commonly seem among organism which from slime layer or capsule. Ex. Kleb. pneumoniae, S. pneumoniae

Rough – colonies are granulated and rough in appearance,
usually produced by mutant strain that lacks surface protein and polysaccharide of freshly isolated wild-type parent organism

Microscopic morphology
• Provide presumptive identification of an organism Bacterial Morphology
• Bacterial cell is a fundamental unit of any living organism • All its functions are genetically controlled and performed by that particular cell structure whether it be physiologic or biochemical • Bacteria and other microorganism are usually transparent, which makes the study of the morphologic detail difficult when they are examined in the natural state • Routinely used to determine: shape arrangement staining reaction

I. Bacterial Shape and Arrangement
 Bacterial Shape • determined by the configuration of the cell wall

detected by brightfield microscopy of stained smear Bacterial Arrangement

is the result of the number of plane division the organism may undergo and how the cell remain attached afterwards  divides only across their short axis

 3 conventional forms :
Spherical (cocci) Rod (bacilli)


Spherical (Cocci)
 Shape:  round like a ball, perfect sphere or globe
 Variations :

1. Ovoid shape - both sides rounded ends are pointed Ex. Streptococcus 2. Lancet-shape - one end is pointed, other end is flat Ex. Pneumococcus 3. Coffee-bean shape - flat on one side, opposite side convex or appear as letter “D” form Ex. Neisseria

1. Singly – occurs as a single spherical cell

2. Chain – “ streptococci” - common among ovoid-form resulting from
one plane division with daughter cells remained attached to one another to form a chain Ex. Streptococcus pyogenes

3. Pairs – “diplococci” - common with lancet-shaped and coffee-bean
shaped spherical resulting from one plane division with daughter cell separating Ex. Streptococcus pneumoniae Neisseria gonorrheae

4. Clusters – “staphylococci”
- common with spherical resulting from many plane division with daughter cell in grape-like agglomeration Ex. Staphylococcus aureus

5. Tetrads – (Packets of 4) - result from 2 plane division with daughter cell
separating from one another to form group of 4 cells Ex. Micrococcus tetragenous

6. Sarcinae – (Packets of 8) - results from many plane division producing
cubical packets of 8 cells Ex. Sarcina lutea

Rods (Bacilli)
cell appears longer than wide or cylindrical form both sides parallel and ends are convex varies in actual form depending on the species divides only across their short axis Variations :

1. Clubbed/drumstick shaped – swollen on one end Ex. Clostridium diphtheriae/C. tetani 2. Corset-shape – both sides swollen, ends concave Ex. Bacillus 3. Fusiform – both sides parallel, ends pointed

flat or anthracis

Arrangements: •Singly – occurs as a single rod •Chain – result from one plane division with
daughter cell remain attached to one another Ex. Bacillus anthracis 3. Palisade – arrangement like fence due to slipping movement of daughter cells (side-by-side) Common among clubbed shaped rods Ex. Mycobacterium tuberculosis 4. Chinese-letter – common with clubbed-shaped rods resulting from a snapping post division movement of the daughter cells (V shape) Ex. Corynebacterium diptheriae 5. Packets of cigarette – arrangement like bundles Ex. Mycobacterium leprae

Intermediate forms Coccobacilli
spherical - when a rod is short & wide/plump - these form is intermediate between a and rod Ex. Haemophilus, Brucella

- a gently curve bacteria (comma-shaped) - it is an intermediate between a rod and a spiral Ex. Vibrio cholerae

bacteria with more than one somatic curve may be regarded as bacillary forms trusted in the form of

a helix no characteristic cell arrangement most occurs singly different specie vary in size, length, rigidity and amplitude of their coils 2 types : 7. Flexible – spirals that can contract and relax & move by creeping movement
Ex. Spirochetes

2. Rigid – spirals that cannot contract and relax & move


rotation or corkscrew-like motion
Ex. Spirillum

when in - whose long axis remains rigid motion Ex. Campylobacter jejuni

– whose long axis bends when in motion
appearance bends Genus Treponema – char. tightly coil w/ cork screw Ex. Trepanema pallidum Genus Leptospira – less tightly coiled w/ sharp hook-like Ex. Leptospira interrogans Genus Borrelia – much less tightly coiled w/c has the appearance of extremely long bacillary pores Ex. Borrelia recurrentis


II. Bacterial size
all linear measurements in microbiology are expressed in

metric units • the basic unit of the metric system is the meter “m” centimeter cm (1/100th of a m) - the largest unit of length used for measuring microorganism micrometer µm - visible only with high powered microscope - unit of measurement most frequently used in microbiology 1µm = 1/1000 of a mm
 Cocci = 0.4-2µm  Bacilli = 0.2-4µm in width by o.5-20µm in length  Spirals = 1-4µm in length

nanometer nm - commonly used to measure virus


Bacterial Staining Reaction

Staining – procedure that applies colored chemicals called dyes to specimen in order to facilitate identification Stains - salts composed of a positive and negative ion, one of which is colored (chromophore – color bearing ion), which imparts a color to cell or cell parts by becoming affixed to them through a chemical reaction Basic (cationic) Dyes - chromophore is the positive ion dye Acid (anionic) Dyes - chromophore is the negative ion dye
Bacteria are slightly negative, so are attracted to the positive chromophore of the BASIC DYE

Preparing smears for staining 1. Smear preparation - depends on the physical state; if in liquid state spread the smear out - Bacteria on slide 2. Air Dry - preserve the morphology of the bacteria - allow the smear to adhere to the slide 3. Bacteria are HEAT FIXED to the slide Heat Fixation - simultaneously kills the specimen and secures it to the slide - preserve various cellular component in a natural state with minimal distortion 4. Stain is applied Staining – coloring the microorganisms with a dye

Types of Staining: 1. Simple Staining
- employs one dye - most common: methylene blue, crystal violet, carbol fuchsin,safranin - sufficient to determine size, shape & arrangement - most cells will stain the same color with the dye used
Positive Staining Appearance of organisms Colored by dye Negative staining Clear and colorless


Not stained (generally white)

Stained (dark gray or black)

2. Differential Staining
- employs the use of more than one dye added in several steps and stained structures are differentiated by color as well as shape - it is based on the relative affinity of different bacterial cells for the stains used - enables microbiologist to differentiate one group from another a) Gram staining - differentiate gram (+) from gram (-) bacteria b) Acidfast staining - differentiate acidfast from nonacidfast bacteria

Hans Christian Gram (1884), a Danish doctor, accidentally

stumbled on a method which still forms the basis for the identification of bacteria; which divided almost all bacteria into two large groups The reagents needed:  Crystal Violet (Primary Stain)  Iodine Solution (Mordant)  Mordant - intensifies the stain or coats a structure to make it thicker and easier to see after it is stained
- Increase the affinity of a stain to the specimen
 Decolorizer (ethanol is a good choice, mixture of acetone &

 Safranin (Counterstain)

Counterstain – gives contrasting color to the primary stain

Gram Staining STEP 1: Make a smear. Mounted and heat fixed. STEP 2: Flood the entire slide with crystal violet (primary stain) for 1min. Then rinse with the water. STEP 3: flood the slide with the iodine solution (mordant) for 1min. Then rinse with water for 5 seconds. The bacteria become deeply stained and appear deep purple in color due to crystal violet-iodine-complex formation

Step 4: addition of the decolorizer, 95% ethanol. Rinse with water. Gram (+) cells : purple dye is retained Gram (-): purple dye is readily removed and appears colorless STEP 5: Flood the slide with the counterstain, safranin Again, rinse with water. Gram (+) cells will incorporate little or no counterstain and will remain purple in appearance Gram (-) bacteria take on a pink/red color

PRINCIPLE:  Gram reaction is based on the structure of the bacterial cell wall  Gram-positive bacteria  the peptidoglycan appears to act as a permeability barrier preventing loss of crystal violet-iodine-complex  When gram-positive bacteria are treated with alcohol, the alcohol causes coagulation and dehydrateion of the thick layer of peptidoglycan resulting in shrinkage of pores preventing CVI-complex from escaping and the bacteria remain deep purple  Reaction to Gram staining is also believed to be asso. With protein complex Magnesium ribonucleate which is absent in Gram (-) org.  Gram Negative bacteria  peptidoglycan is very thin in gram (-) bacteria and has larger pores  Alcohol readily penetrates the lipid­rich outer layer of the cell wall and extracts enough lipid thus increasing the porosity further  alcohol more readily removes the deep purple CVI-complex

Divides bacteria into 2 groups  Gram (+) : violet  Gram (-) : red

Dictome of Gram Staining All COCCI are Gram Positive except Neisseria group, Moraxella (Branhamella) catarrhalis and Veilonella
All BACILLI are Gram Negative except the acid

fast organisms (Mycobacterium, Nocardia) , Sporeformers (Bacillus, Clostridum) and Corynebacterium species

Acid Fast Staining
Acid-fast stain is a useful differential staining procedure

that specifically stains all members of the genera mycobacteria The walls of certain bacteria contain long chain fatty acids (mycolic acid) lending the property of resistance to decolorization of basic dyes by acid alcohol; thus called “acid fast” The high lipid and wax content of the mycobacterial cell walls is thought to be the reason for such impermeability 2 methods
 Ziehl-Neelsen method

The procedure utilizes heat and phenol (carbolic acid) to help the penetration of the dye, carbol fuchsin, to the inside of mycobacterial cells, which are impermeable to basic dyes in routine stains like in Gram staining  Cold Kinyoun technique

Divides bacteria into 2 groups
 Acid - Fast organism: red  Non Acid – Fast organism: blue

The reagents needed

1. Primary stain: Carbol fuchsin 2. Decolorizer: Acid Alcohol 3. Counterstain: Methylene Blue

Acid - Fast Staining (Ziehl-Neelsen
STEP 1: Make a smear. Mounted and heat fixed STEP 2: Flood the entire slide with Carbol Fuchsin. STEP 3: Using a Bunsen burner, heat the slides slowly until they are steaming. Acid fast organisms have a very hydrophobic surface which resist entry of dyes. Heat is used to enhance penetration and retention of dye Maintain steaming for 5 minutes by using low or intermittent heat (i.e. by occasionally passing the flame from the Bunsen burner over the slides) Then rinse the slide with water. STEP 4: Flood the slide with 3% acid-alcohol and allow to decolorize for 5 minutes. Throughout the 5 minutes, continue to flood the slides with 3% acid-alcohol until the slides are clear of stain visible to the naked eye. Rinse the slide thoroughly with water and then drain any excess from the slides. STEP 5: Flood with the counterstain, Methylene Blue Keep the counterstain on the slides for 1 minute. Rinse with water.

3. Special Staining
- used to color and isolate specific structure of a microorganism like capsule, flagella, inclusion granule, endospore and etc. Positive Staining Negative staining



Biochemical Test
various species of organism exhibits characteristic

pattern of substrate utilization, metabolic product formation and sugar fermentation
 Enzyme based test – based on its reaction with a


Catalase, oxidase, indole, urease

 Reactions in glucose fermentation broth  Reactions in lactose fermenation broth  Starch hydrolysis of test strains  Nitrate Broth reactions

60% of common pathogens can be identified by

metabolic test

Serological procedure
Antigen and antibody determination Serological Tests
 Use group specific antiserum isolated from the plasma

of animals that have been sensitized to the organism  The antiserum contains antibody proteins that react with antigens on the unknown organism. Procedures: agglutination, precipitation test, hemagglutination inhibition, complement fixation, ELISA, RIA, Western blot assay Advantages:  Highly specific  Does not usually require the organism to be isolated into pure culture  Can be used to identify organisms that can’t be grown on medium

Antibiotic sensitivity
antibiotic sensitivity is a term used to describe the

susceptibility of bacteria to antibiotics Antibiotic susceptibility testing (AST) is usually carried out to determine which antibiotic will be most successful in treating a bacterial infection in vivo Methods of testing:  Broth dilution  The lower the dilution, the greater the antibiotic content  Agar dilution  Disk diffusion  the Kirby-Bauer test for antibiotic susceptibility, called the disc diffusion test, is a standard that has been used for years

 The bacterium is swabbed on the agar

and the antibiotic discs are placed on top
 The antibiotic diffuses from the disc into

the agar in decreasing amounts the further it is away from the disc
 Bacteria are not able to grow around antibiotics

to which they are sensitive
 If the organism is killed or inhibited by the

concentration of the antibiotic, there will be NO growth in the immediate area around the disc: called the zone of inhibition The zone sizes are looked up on a standardized chart to give a result of sensititive, resistant, or intermediate
 Many charts have a corresponding column that also gives

the MIC (minimal inhibitory concentration) for that drug