MICLAB-070 Identification of Microorganisms to Genus and Species Level

Aseptic Technique must be used when performing any Microbiological Identification procedures.

All tests using live organisms should be performed in a Biological Safety Cabinet.

All disposable contaminated material should be disposed of in the correct biohazard bins or decontaminating solution.

All non-disposable contaminated material should be autoclaved before cleaning.

Care should be taken when using the Biohazard Cabinet, especially when using gas or when UV light sterilisation is in process.

Decontamination agents can be irritating to the eyes and skin.

Safety glasses and gloves are to be worn when using IPA/Solvent. 

1. Introduction

Bacteria that will require identification (ID) to at least genus level include organisms isolated from the manufacturing environment, personnel, in-process and finished products, plant water and other miscellaneous sources.  SOP’s detailing the microbiological testing procedures for each of these samples will indicate the required level of ID of recovered organisms. The following sections detail the procedures for the preliminary ID of microorganisms. Further ID to species level is to be conducted for conformation.

2. Administration and Preparation of Isolates

2.1.         Classification of Test Samples

Microbiological tests that have recovered organisms, which require ID, are to be classified into seven ID categories according to the nature of the test sample. These categories are listed in Table 1.

Table 1:  ID Categories for Microbiological Tests

2.2.         Unique Sample Identifier

Each microbial isolate requiring ID will be assigned a Unique Sample Identifier (USI) relative to the category to which the sample belongs.  The isolate identifier is a 6-digit number comprised of the Category Prefix (Table 1), an Isolate Number and the number of differing isolate types recovered from the test sample.

The USI distinguishes each microbial isolate requiring ID and provides a point of reference between the test result and the associated ID results of organisms recovered from the test.

The following example describes how the USI is created.

Example:  Following the specified incubation period, an air sample results in the growth of 2 colony-forming units (CFU).  These CFU are of differing macroscopic morphology i.e. they are two different isolate types.  The result is an OOL (Out of Limit) result, thus the organisms isolated from the sample require ID.

The USI assigned is as follows:

FIGURE

2.3.         Preparing Isolates for ID

2.3.1.     For example where the USI is for e.g. 010326/3 – the number of isolate types is 3.  These isolate types will be distinguished from one another using the following nomenclature; Category prefix, followed by the Isolate number, followed by the isolate type number i.e. 010326-1, 010326-2 and 010326-3.  This is further illustrated in the following example:

The allocated USI for a given test sample is as follows:

010326/3

3 isolate types were recovered from this sample.

These isolate types will have a defined isolate type number as follows:

Isolate Type 1:  010326-1

Isolate Type 2: 010326-2

Isolate Type 3: 010326-3

2.3.2.     Define the isolate types in the test sample using the given nomenclature.

2.3.3.     Fill out an Isolate ID Record form appropriate to the ID Category of the test sample.

2.3.4.     Record the details as follows:

Sample ID # – this is the USI

Total CFU/Growth – the total number of colonies recovered from the test sample OR

Growth/No Growth in broth

Sample Details – including sample/test date, location, batch #, inspection lot number (where applicable), associated Deviation Report (if any) etc.

Sample Type/Machine/Environmental Grade/Water Type – to be specified where applicable

Isolate ID # – isolate type number assigned to each individual isolate type

Macroscopic Description  – morphological description of the isolate; size, colour, shape, margin, lustre etc. (see Appendix 7.1).

CFU – number of colony forming units bearing the same macroscopic description.

2.3.5.     Streak each isolate type onto the respective TSA plate labelled with the Isolate type marking.  Use the following procedure to ensure growth of single, pure, well-isolated colonies.

2.3.6.     Flame a wire loop until red-hot by passing through the hottest part of a Bunsen burner flame OR take a sterile disposable loop.  If using a wire loop, allow the loop to cool before proceeding (to cool the loop quickly touch the loop against the agar of the TSA plate to be used).

2.3.7.     With the sterile loop, lightly touch the colony to be streaked OR take one loopful of a broth culture from the test sample and immediately streak the growth onto the TSA plate creating a primary inoculation well (Fig. 1a).

2.3.8.     Flame the wire loop and allow to cool OR take a fresh sterile disposable loop and streak 4-7 parallel streak lines from the primary well (Fig. 1b).

2.3.9.     Flame the wire loop and allow to cool OR take a fresh sterile disposable loop and streak 4 parallel streak lines from those streaked in step 1.4.6.3 (Fig. 1c).

2.3.10.   Flame the wire loop and allow to cool OR take a fresh sterile disposable loop and streak 4 parallel streak lines from those streaked in step 1.4.6.4 (Fig. 1d).

2.3.11.   Flame the wire loop and allow to cool OR take a fresh sterile disposable loop and streak 4 parallel streak lines from those streaked in step 1.4.6.5 (Fig. 1e).  Extend the last streak line into the centre of the plate. Note: A fresh sterile loop must be used for each new set of streak lines OR a wire loop must be flamed and cooled.  Using a sterile loop facilitates the dilution of organisms with each set of streak lines enabling the growth of single colonies.

Figure 1: Plate Streaking for Single Colonies

2.3.11.1.  Incubate all plates at 30-35ºC for 24 hours.  Slow growing and stressed organisms may require longer incubation.  Yeasts may require incubation for up to 7 days.  Store the original test sample plate/broth labelled with the assigned USI at 2-8ºC until all ID work is complete.  Some test samples will require a longer period of retention under these conditions (see Table 2).  Ensure the integrity of all original test samples is preserved during storage.

Table 2:  Test Sample Retention Periods

2.3.11.2.  Place the Isolate ID Record form for the test sample in the In Process tray in the ID laboratory.

3. ID of Isolates

Note: Before proceeding with the following ID procedures visually inspect all streaked isolate cultures for purity and the presence of single, isolated colonies.  If the culture appears mixed, re-streak the isolate for purity.

Only single, isolated colonies are to be used for the following characterisation tests.  Purity cannot be visually confirmed in areas of heavy or confluent growth.

Note: If a particular isolate cannot be cultured on solid media, inoculate the organism into 10mL TSB to resuscitate and incubate at 30-35ºC for 24-48 hrs. The resuscitated broth may be gram stained and then plated onto TSA (if required). 

If the subculture of ANY isolate proves to be unsuccessful, The Microbiology Manager must be informed immediately.

Note: Appendices 7.3 to 7.6 will assist in the identification of isolates.  These should be used as a guide.

3.1.         The Gram Stain

Organisms are classified according to their gram stain reaction.  The gram stain is the primary and most critical step in the identification process.

Gram-positive bacteria have thicker and denser peptidoglycan layers in their cell wall.  Iodine penetrates the cell wall in these bacteria and forms a Crystal Violet/Iodine (CV/I) complex inhibiting diffusion of this complex through the cell wall during decolourisation.  Gram-positive bacteria must have an intact cell wall to produce a positive reaction.  The cell wall of gram-positive bacteria will deteriorate as the plate culture ages; therefore cultures must be fresh when conducting the Gram Stain.

Gram-negative cells do not retain the CV/I complex, due to a far less complex peptidoglycan layer in the cell wall are stained using a counter stain.  Neutral red, Safranin or Carbol Fuchsin may be used as the counterstain.

3.1.1.     Prepare a smear:

Plate Culture – Using a sterile loop, lightly touch the colony to be examined and emulsify into a drop of sterile water on a glass slide.

Broth Culture – Using a sterile loop, remove a loopful of culture and smear directly onto a glass slide.

Note:  Avoid picking up too much growth as this will result in a heavy smear, which will not stain effectively and cannot be examined with accuracy.

Note:  You may wish to vortex a broth culture prior to creating a smear as this will ensure an even mix of cells in the culture.  As the broth is a liquid culture, water is not required to create the smear.

3.1.2.     Allow the smear to air dry and heat fix the cells to the slide by passing the slide through the hottest part of the flame three times.  Allow to cool.

3.1.3.     Flood the slide with Crystal Violet solution and stand for 30 seconds (Fig. 2A).

3.1.4.     Rinse off Crystal Violet solution with water and tilt the slide to drain excess water.

3.1.5.     Flood the slide with 1% Lugol’s Iodine and stand for 30 seconds (Fig. 2B).

3.1.6.     Rinse off Iodine with water and tilt the slide to drain excess water.

3.1.7.     Flood the slide with Decolourising solution and immediately rinse the slide with running water after 3 seconds (Fig. 2C).  Tilt the slide to drain excess water.

Note:  Prolonged contact with decolourising solution will result in over-decolourisation of cells and a false gram reaction.  Ensure the contact time is limited to 3 seconds only.

3.1.8.     Flood the slide with the desired counterstaining solution.  Leave to act for up to 2 minutes (Fig. 2D).

3.1.9.     Rinse with water and blot dry using a paper towel.  Do not rub.

3.1.10.   Examine the smears under Oil Immersion.

3.1.11.   Record the cellular arrangement (see Appendix 7.2) and Gram stain result of the organism on the Isolate Identification Record form.

3.1.12.   Interpretation

Gram-positive                    Gram-positive organisms stain purple

Gram-negative                   Gram-negative organisms stain pink/red.

3.1.13.   Quality Control

3.1.13.1.  Gram-positive and Gram-negative reference cultures are to be used for quality control of the gram staining procedure and reagents.

Gram Positive                 Staphylococcus aureus ATCC 6538

Gram Negative                Escherichia coli ATCC 8739

3.2.         Technical Information

Some Gram-positive bacteria may appear Gram-negative in whole or in part eg. some strains of Gram-variable Bacillus and Clostridium.  Yeasts will appear “Gram-positive” and will be visibly much larger than a prokaryotic bacterial cell.

All Gram-variable and suspected over or under-decolourised organisms are to be subject to a KOH test (see section 3.3).

Figure 2: The Gram Stain

3.3.         The Potassium Hydroxide (KOH) test

Known Gram-variable organisms and organisms which have lost some of their cell wall integrity (due to an ageing culture), appear Gram-negative on staining, resulting in possible misidentification.

The KOH test (also known as the “String Test”) aids in the differentiation of Gram-positive bacteria from Gram-negative organisms.

In the presence of potassium hydroxide, Gram-negative cell walls are broken down, releasing viscid chromosomal material, which causes the bacterial suspension to become thick and stringy.  Most Gram-positive organisms remain unaffected.

Note:  The KOH test can only be performed using colonies grown on solid medium. The KOH test is to be performed on all non spore-forming rods and where the Gram reaction of any organism cannot be determined due to Gram variability or poor staining. The KOH test is positive for 100% of all Gram-negative organisms but only 97% negative for all Gram-positive organisms. A negative result can only be obtained from a Gram-positive organism; a positive result can be obtained from all Gram-negative organisms and some Gram-positive organisms.

3.3.1.     Place one drop of 3% KOH solution on a clean microscope slide.

3.3.2.     Using a sterile loop pick up 2-3 colonies and emulsify in the KOH to make a dense suspension.  The suspension should appear cloudy if a sufficient number of colonies have been picked.

3.3.3.     Mix the suspension continuously for up to 60 seconds, and then gently pull the loop away from the suspension.  If the organism is Gram-negative the suspension will form a string which adheres to the loop and stretches from the slide (Fig. 3).

Note:  A positive result may be evident in as little as 5 seconds.

3.3.4.     Record the KOH result of the organism on the Isolate ID Record form.

Figure 3: Positive KOH Test

3.3.5.     Interpretation

Positive result: Gram-negative organisms become thick and stringy and form long strands

Negative result: Organism is Gram-positive and remains unaltered

3.3.6.     Quality Control

Gram-positive and Gram-negative reference cultures are to be used for quality control of the KOH reagent at each use.  Do not use the test if reactions with the control organisms are incorrect.

QC organisms

Positive Control           Escherichia coli ATCC 8739

Negative Control          Staphylococcus aureus ATCC 6538

3.4.         The Catalase Test

This test detects the catalase enzyme present in most cytochrome-containing aerobic and anaerobic bacteria.  The catalase enzyme decomposes Hydrogen Peroxide (H₂0₂) to release Oxygen and Water.

Note: Hydrogen Peroxide solution should be stored at 2-8ºC.  The solution is light sensitive and should be stored appropriately avoiding any undue exposure to light.

The Catalase test is to be conducted on all Gram-positive bacteria.

3.4.1.     Place a drop of 3% H₂0₂ solution on a clean microscope slide.

3.4.2.     Using a sterile loop, pick up a single isolated colony and immerse the loop and the adhering colony mass into the drop of H₂0₂ solution.

3.4.3.     Look for vigorous bubbling occurring within 10 seconds.

3.4.4.     Record the catalase result of the organism on the Isolate ID Record form.

Figure 4: Catalase Reactions

3.4.5.     Interpretation

Positive result: Vigorous bubbling indicating the presence of catalase enzyme converting H₂0₂ to oxygen and water

Negative result: No bubbling

3.4.6.     Quality Control

Control organisms are to be tested with each use, as H₂0₂ is unstable. Do not use the test if reactions with the control organisms are incorrect.

QC organisms

Positive Control           Staphylococcus aureus ATCC 6538

Negative Control          Enterococcus casseliflavus ATCC 700327

3.4.7.     Technical Information

The enzyme is present in viable cultures only.  Do not perform on cultures over 24 hours old as this may result in a false negative reaction.

Anaerobic cultures should be exposed to air for 30 minutes prior to testing.

3.5.         The Coagulase Test

Members of the genus Staphylococcus are differentiated by the ability to clot plasma by the action of the enzyme coagulase.

Coagulase exists in two forms:

Bound (Clumping Factor) – enzyme is bound to the cell wall.  Enzyme absorbs fibrinogen from the plasma and alters it so it precipitates on the Staphylococci, causing them to clump – resulting in cell agglutination.

Free – enzyme is liberated by the cell wall and reacts with a substance in plasma to form a fibrin clot.

Staphytect Plus is a latex slide agglutination test for the detection of clumping factor, Protein A and certain polysaccharides found in methicillin resistant S.aureus (MRSA) from those staphylococci that do not possess these properties.  It is much more sensitive than the traditional slide coagulase test due to its ability to detect Protein A and capsular polysaccharide.

Note: The Staphytect Kit must be stored at 4ºC away from direct sunlight or heat sources.  Ensure the caps are securely fitted after each use to prevent contamination and drying out of the reagent.

3.5.1.     Bring the latex reagents to room temperature.

3.5.2.     Vortex the latex reagent for 10 seconds and dispense one drop of test latex into one circle on the reaction card and one drop of control latex into another circle on the reaction card.

3.5.3.     Using a sterile disposable loop, pick up 5 single isolated colonies and immerse the loop and the adhering colony mass into the Control latex reagent. Mix and spread to cover the entire circle.

3.5.4.     Using a fresh sterile disposable loop repeat in the same way with the Test Latex.

3.5.5.     Pick up and rock the card for up to 20 seconds and observe for agglutination.

3.5.6.     Record the Coagulase result of the organism on the Isolate ID Record form.

Figure 5: The Coagulase Test

3.5.7.     Interpretation

Positive result:      A result is positive if agglutination of the blue latex particles occurs within 20 seconds.  This presumptively identifies the strain as Staphylococcus aureus.

Negative result:     No agglutination occurs and a smooth blue suspension remains after 20 seconds in the test circle.  This presumptively identifies the strain as a non S.aureus.

Equivocal result:   Slight graininess of the test latex accompanied by no change in the appearance of the control latex should be interpreted as an equivocal result.  Strains should be re-tested following subculture.

Uninterpretable result:      The test is uninterpretable if the control reagent shows agglutination.  This indicates that the culture causes autoagglutination.

3.5.8.     Quality Control

Control organisms are to be tested with each use.  Do not use the test if reactions with the control organisms are incorrect.

QC organisms

Positive Control           Staphylococcus aureus ATCC 6538

Negative Control          Staphylococcus saprophyticus BAA-750

3.5.9.     Technical Information

The tendency of isolated colonies to cause autoagglutination increases with incubation times over 36 hours.

Only Staphylococci should be tested using the Latex test, as species such as Escherichia coli are able to agglutinate latex particles non-specifically.

All positive, presumptive S.aureus cultures are to be confirmed further.

3.6.         Sporulation and The Spore Stain

Members of the genus Bacillus have the ability to produce endospores under unfavourable growth conditions.  The conditions under which sporulation takes place determine the spore characteristics in terms of chemical and/or heat resistance.  Sporulation is not always evident in fresh cultures grown on general-purpose media, as the conditions may not stimulate the formation of a spore.

It is of high importance that isolates collected from Filled Container Bioburdens (FCB) do not present any Gram Positive Sporing Rods. Any test samples presenting this type of growth will need a D-value performed. Refer to MICLAB 075.

To ensure that all GPRs are identified with accuracy, spore formation must be determined. All Gram-positive rods collected from FCBs, which do not appear to be spore formers after culturing using TSA, are to be cultured using Sporulation Agar (SA).  A spore stain may then be used for the demonstration of spores.

Note: SA is a specialised medium used to stimulate bacterial spore generation by Bacillus spp.  The medium contains peptones as a source of nitrogen, beef extract and yeast extract provide essential vitamins and growth factors for metabolism, while a low level of glucose provides a limiting source of carbon. The limited carbon source coupled with the addition of Manganese Sulphate stimulates sporulation.

3.6.1.     Streak the isolate onto SA and incubate at 35-37ºC for 3-5 days.

3.6.2.     Prepare a smear and heat fix the cells to the slide by passing the slide through the hottest part of the flame three times. Allow to cool.

3.6.3.     Conduct a Spore Stain to observe spore formation. See MICLAB 065.

3.6.4.     Examine the smear under Oil Immersion.

3.6.5.     Interpretation

Positive                 Spores stain green

Negative                Vegetative cells stain pink/red

Note: If sporulation is evident following culture on SA the organism is presumptively identified as a Bacillus spp.

If sporulation is not evident in the culture following 5 days incubation, the organism is presumptively identified as a Non-sporing Gram-positive rod.

3.6.6.     Quality Control

Spore forming and non-spore forming reference cultures are to be used for quality control of the spore staining procedure and reagents.

QC organisms

Positive                        Bacillus subtilis ATCC 6633

Negative                       Escherichia coli ATCC 8739

4. Quality Control (QC)

4.1.         QC and Traceability of Reagents/Kits

4.1.1.     For traceability purposes, the batch number and/or expiry date of all reagents used in characterization tests are to be logged in the ID Reagents Logbook on a daily basis.

4.1.2.     Reagents are to be challenged at each use (i.e. once daily) using the stated QC organisms (see Table 3).  Staining reagents are to be challenged upon opening, using the stated QC organisms.  They may then be verified for use.

4.1.3.     The challenge organisms are to be no more than 24 hours old.  Ensure a pure, fresh culture is always available for QC.

4.1.4.     Record the QC results for all reagents in the ID Reagents Logbook.

Table 3:Quick Reference Table of QC Organisms for Regents/Kits

5. Documentation of Results

5.1.         Results Recording and Further ID

5.1.1.     When complete, the results of all characterisation tests conducted for each isolate are to be recorded on the Isolate ID Record forms.  Record any additional information in the Additional Comments field.

5.1.2      Determine whether further ID of the isolate is required.  This will be stated in the SOP relevant to the test sample from which the organism was recovered.

5.1.2.     If the organism does not require any further ID, indicate this on the Isolate ID Record form and initial and date against all information recorded for the isolate.  The subcultured isolate and original test sample may be discarded when all ID work and documentation is complete (check the retention requirements of the test sample and associated isolates before disposal – see Table 2).

5.1.3.     All completed Isolate ID Record forms are to be reviewed and approved upon completion.  Once approved, all ID results are to be entered into the Sample ID Number Allocation spreadsheet.

5.1.4.     The approved Isolate ID Record forms are to be filed in the Completed Identifications File.  A separate file should exist for each ID category (see Table 1).  File all forms in numerical sequence according to the assigned USI.  This will ensure quick retrieval of ID records when required.

6. Monitoring Trends

6.1.         Trending of Microbial ID Results

6.1.1.     Where appropriate, trending of microbial isolates will be conducted on a routine basis in conjunction with trending of test results for the different sample types; including Environmental and Personnel monitoring samples, Settle plates, Disinfectant samples, Water, Non-sterile & Raw Material samples and Sterility test failures. The Microbial ID “Sample ID Number Allocation” spreadsheet can be used to filter data specific to different sample types to aid in trending.

6.1.2.     Trending of microbial isolates should include number, frequency and types of different microorganisms and from various sample types. Microbial ID trending should also include a review of the most commonly identified isolates from various sample types.

7. Appendices

7.1.         Colony Morphology

Describe all of the following colony characteristics:

SIZE OF COLONY – in mm

CHROMOGENESIS (Pigmentation): white, off white, cream, pink, brown etc.

SHAPE OF COLONY (Fig. 6) – circular (if less than 1mm in diameter shape is punctiform), irregular, filamentous or rhizoid

EDGE/MARGIN OF COLONY (Fig. 6)– entire, undulate, filiform, curled or lobate

OPACITY OF COLONY – transparent, opaque, translucent or iridescent (changing colours in reflected light)

ELEVATION OF COLONY (Fig. 6) – raised, convex, flat, umbonate or crateriform

SURFACE OF COLONY – smooth, glistening, rough, dull (opposite of glistening) or rugose (wrinkled)

CONSISTENCY: butyrous (buttery), viscid (sticks to loop), brittle/friable (dry, breaks apart), or mucoid

Figure 6: Form, Elevation and Margin of Colonies

7.2.         Cellular Shape and Arrangement

Most bacteria that will be isolated and examined in the laboratory will appear in one of two basic shapes – coccus or rod/bacillus.

Coccus – The cocci are spherical or oval bacteria having one of several distinct arrangements based on their planes of division (Fig. 7A).

Division in one plane produces either a diplococcus or streptococcus arrangement

Division in two planes produces a tetrad arrangement

Division in random planes produces a staphylococcus arrangement

Rod/Bacillus – Bacilli are rod-shaped bacteria. Bacilli all divide in one plane producing a bacillus, streptobacillus, or coccobacillus arrangement (Fig 7B).

Pleomorphic rods of the genus Corynebacterium also divide in one plane, however they possess a characteristic v-shaped arrangement known as a palisade (Fig. 7B).  This is due to a snapping movement, which occurs immediately after cell division, which brings the cells into this arrangement.

Figure 7: Cellular Arrangements

7.3.        ID Flowchart: Gram Positive Cocci

7.4.        ID Flowchart: Gram Positive Rods

7.5.        ID Flowchart: Gram Negative Rods

7.6.        ID Flowchart: Yeasts

7.7.         Gram Positive Cocci

7.7.1.     Staphylococcus species

Figure 8: Gram Stain of Staphylococcus cells

7.7.2.     General Information

Gram-positive, non-motile, non-sporing cocci occurring singly, in pairs and in irregular clusters

Colonies are opaque and may be white, cream and occasionally yellow and orange

Optimum growth temperature is between 30-37ºC

They are facultative anaerobes and have a fermentative metabolism

Catalase positive and oxidase negative

Usually able to grow in 10% Sodium Chloride

Staphylococcus aureus is a primary pathogen.  Produces virulence factors such as Protein A, capsular polysaccharide and a toxin

Coagulase positive*

*(S.aureus is not the only coagulase positive Staphylococcus – see Table 5 below)

7.7.2.1. Sources – Normal flora of the following sites:

Table 5:  Commonly isolated Coagulase positive and negative Staphylococcus Species

7.7.3.     Micrococcus species

Figure 09: Gram Stain of Micrococcus cells

7.7.4.     General Information

Micrococcus species are strictly aerobic.

Micrococcus luteus produces yellow colonies.

Cells are large Gram-positive cocci arranged in tetrads.

Catalase positive.

Optimum growth temperature is between 30-37ºC.

Sources – Environmental – Soil, Water; Human – Skin

7.7.5.     Streptococcus species

Figure 10: Gram Stain of Streptococcus cells

7.7.6.     General Information

Streptococcus species are facultatively anaerobic.

Cells are Gram-positive non-motile cocci arranged in chains or pairs.

Catalase negative.

Haemolysis on Blood agar.

Optimum growth temperature is between 30-37ºC.

Sources – Normal flora of the following sites:

7.8.         Gram Positive Rods – Sporing

7.8.1.     Bacillus species

Figure 11: Gram Stain of Bacillus cells

7.8.2.     General Information

The genus Bacillus comprises in excess of 60 species, commonly found in the environment and as laboratory contaminants.

Bacillus spp. are Gram-positive rods arranged in pairs or chains with rounded or square ends and usually have a single endospore

The endospores are generally oval and are very resistant to adverse conditions

Colonies are flat and irregular, often with numerous undulated outgrowths of long filamentous chains of bacilli

Aerobic or facultatively anaerobic and most species are motile by peritrichous flagella

Fermentative metabolism.

Oxidase positive (most species)

Catalase positive (most species)

Sources – Environmental – Soil, Contaminated Food, Rice (B.cereus); Animal – Animals and Animal Products

Bacillus species can be broadly divided in three groups based on the morphology of the spore and the sporangium.

1. Gram-positive, produce central or terminal ellipsoidal spores that do not distend the sporangium

*Bacillus thuringiensis produces cuboid or diamond shaped parasporal crystals in cultures on sporulation agar or nutrient agar incubated for at least 2 days.  Crystals are demonstrated by staining with malachite green.

2. Gram-variable with ellipsoidal spores and swollen sporangia

3. Gram-variable, sporangia swollen with terminal or subterminal spores

B.sphaericus

7.8.3.     Clostridium species

Figure 12: Gram Stain of Clostridium cells

7.8.4.     General Information

Clostridia are anaerobic, Gram-positive or Gram-variable, spore forming, bacteria.

Members of this genus resemble large, straight or slightly curved rods with rounded ends often arranged in pairs or short chains.

All the members of the genus, except Clostridium perfringens, are motile with peritrichous flagella and form oval or spherical endospores that may distend the cell.

They are usually Catalase negative.

Optimal growth conditions – incubation under anaerobic conditions at 35°C – 37°C for 40-48h.

Sources – Environmental – Soil, Contaminated Water; Human – Gastrointestinal tract, Female Genital Tract (C.perfringens)

7.9.         Gram Positive Rods – Non-sporing

7.9.1.     Lactobacillus species

Figure 13: Gram Stain of Lactobacillus cells

7.9.2.     General Information

Gram-positive large rods, non-spore forming, anaerobic or microaerophilic, occur singly or in pairs.

Convert Lactose and other sugars to Lactic Acid.

Sources – Normal flora of the following sites of Humans and Animals:

7.9.3.     Corynebacterium species

 Figure 14: Gram Stain of Corynebacterium cells

7.9.4.     General Information

Corynebacterium species are Gram-positive, pleomorphic, non-spore forming and slightly curved rods with tapered or clubbed ends.

Cells may occur singly or in pairs, often in a “V” formation due to a snapping mechanism occurring after cell division. Cells will usually stain weakly and unevenly giving a beaded appearance.  This is due to old cells storing inorganic phosphates in their cell wall, which appear as metachromatic granules when stained.

Aerobic or Facultatively anaerobic.

Catalase positive.

Sources – Environmental – Soil, Water; Human – Skin, Nasopharynx

7.10.      Gram Negative Organisms (Non-glucose fermenters)

7.10.1.   Acinetobacter species

Figure 15: Gram Stain of Acinetobacter cells

7.10.2.   General Information

Species of the genus Acinetobacter are strictly aerobic non-fermentative, non-motile Gram-negative bacilli. They show predominantly a coccobacillary morphology on nonselective agar and appear in pairs under the microscope.

They may not readily decolourise on Gram staining and demonstrate variable stain retention, along with pleomorphic variations in cell size and arrangement.

Many strains are encapsulated.

Colonies are normally smooth, sometimes mucoid, pale yellow to greyish-white and some environmental strains may produce a diffusible brown pigment.

Most strains have an optimum growth temperature of 30°C – 35°C and grow well at 37°C although some are unable to grow at 37°C.

Catalase positive.

Oxidase negative.

Sources – Environmental – Soil, Water

7.10.3.   Pseudomonas species

Figure 16: Gram Stain of Pseudomonas cells

7.10.4.   General Information

Species of the genus Pseudomonas are strictly aerobic, glucose non-fermenting Gram-negative rods.  Motile by means of a single polar flagellum.

Growth temperatures range from 5°C – 42°C.  Optimum growth temperature is 37ºC.

Oxidase positive

Catalase positive

P. aeruginosa is one of the only members of the species that can sustain growth at 42ºC.  The characteristic blue-green appearance of cultures is due to the production and mixture of pigments -pyocyanin (blue) and pyoverdin (fluorescein, yellow).

Some strains produce other pigments, such as pyorubin (red) or pyomelanin (brown).

P. putida and P. fluorescens are members of the fluorescent group of pseudomonads. Unlike P. aeruginosa they are unable to grow at 42°C and do not produce pyocyanin.

P.stutzeri produces smooth, intermediate and rough colonies (sometimes yellow

pigmented) on non-selective agar. The latter can resemble colonies of Burkholderia

pseudomallei or Bacillus species.

P. alcaligenes and P. pseudoalcaligenes are both non-pigmented.

Sources – Environmental –Water, Soil; Human – Gastrointestinal tract (Normal flora in a small percentage of healthy individuals).

7.10.5.   Other commonly isolated Non-fermenting Gram-negative rods include:

Achromobacter (Alcaligenes) xylosoxidans

Alcaligenes xylosoxidans was reclassified as Achromobacter xylosoxidans in 1998. It is both catalase- and oxidase positive.

Alcaligenes species

Colonies have a thin, spreading irregular edge. It is catalase negative, oxidase positive and motile.

Brevundimonas species

Brevundimonas vesicularis and Brevundimonas diminuta grow slowly on ordinary nutrient media.  It forms a carotenoid pigment that produces yellow or orange colonies.

Elizabethkingia species

Elizabethkingia (formerly Chryseobacterium) meningosepticum, is the species of Elizabethkingia most often associated with serious infection. E. meningosepticum is non-motile and oxidase positive. E. indologenes is also non-motile and oxidase positive.

Comamonas species

It is motile, oxidase and catalase positive.

Methylobacterium species

The organism is oxidase positive and motile, but both of these characteristics may be weak. Methylobacterium species are Gram-negative but may stain poorly or show variable results. It has a characteristic microscopic appearance because individual cells contain large, non-staining vacuoles.

Ochrobactrum species

Colonies appear circular, low convex, smooth, and shining. Mucoid colonies may be produced on some media.

Ralstonia species

Ralstonia pickettii (formerly Burkholderia pickettii) is non-pigmented, oxidase-positive, and will grow at 41°C.

Shewanella species

Shewanella putrefaciens is oxidase-positive and motile.

Sphingobacterium species

They are oxidase-positive and non-motile. Colonies produce yellow pigment.

Stenotrophomonas species

Catalase positive and Oxidase negative.

7.11.       Gram Negative Organisms (Glucose fermenters)

Figure 17: Gram Stain of Escherichia coli cells

7.11.1.   General Information

E. coli is the head of the large bacterial family, Enterobacteriaceae, the enteric bacteria, which are facultatively anaerobic Gram-negative rods.

A number of genera within the family are human intestinal pathogens (e.g. Salmonella, Shigella, Yersinia). Several others are normal colonists of the human gastrointestinal tract (e.g. Escherichia, Enterobacter, Klebsiella), but these bacteria, as well, may occasionally be associated with diseases of humans.

The enterics ferment glucose producing acid and gas

Optimum growth temperature is 37ºC.

Oxidase negative

Produce peritrichous flagella.

Sources – Human/Animals – Gastrointestinal tract, faecal material

8. Summary of Changes