Specimen Collection and Transport

The most important aspects of microbiological testing are collection of the right specimen and transport of the specimen to lab in a manner that insure the reliability of the diagnostic procedure (e.g., culture, microscopy and antigen or antibody tests).



As a general guideline for all specimens the following consideration should be kept in mind:

 Specimens should always be considered infectious. Therefore, gloves should always be worn when handling specimens.

 Specimens should be collected from the areas where organisms are present. For example, diagnosis of a lower respiratory tract infection requires collection of material from that site (e.g., sputum) and not from the mouth (e.g., saliva)  The quantity of specimen collected must be sufficient to ensure that all requested tests (culture, microscopy, antigen tests ,nucleic acid probes and amplification) can be performed properly.

 Transport of specimens should maintain the viability of etiologic agent (if culture is performed) and prevent overgrowth with contaminating organisms.  Specimens should always be transported in a leak-proof container inserted in a leak proof plastic bag.

 for shipement of speciemen or isolates, refer to the International Air Transport Association (IATA) Dangerous Goods Regulation ( http://www.IATA.org/dangerousgoods/index ), the U.S. Department of Transportation ( http://hazmat.dot.gov/rules.htm

) and the international Civil Aviation Organization (ICAO). Each specimen type requires specific packing and handling procedures.

The Classification and Identification of Bacteria of Medical Importance

Gram negative cell wall Gram positive cell wall

One of the most important techniques in bacteriology is the Gram stain, first described by Hans-Christian Gram in 1884.

Gram stain

It allows the differentiation of bacteria into two groups:

Gram positive

bacteria can retain a complex made between crystal violet and iodine when decolourised with acetone, whereas

Gram-negative

bacteria are decolourised on treatment with acetone.

the Gram stain enables the shape of bacterial cells to be observed easily. Bacteria fall into two major classes: rod shaped bacteria are known as '

bacilli

' (singular: 'bacillus') and round or roundish bacteria are known as '

cocci

' (singular: 'coccus'). This property reveals fundamental differences in the cell envelope between the two groups. Gram-positive bacteria have many layers of peptidoglycan in their cell wall; Gram-negative bacteria have only one or two peptidoglycan layers but, additionally, they have an outer membrane. These differences have important consequences. For example, certain antibiotics cannot penetrate the outer membrane of Gram-negative bacteria, which are intrinsically resistant to these drugs as a consequence.

Micrococcus luteus Staphylococcus aureus Bacillus subtilis

E. coli

Serratia marcescens

Performance standards for stains

Stain

Ziehl-Neelsen Giemsa Acridine orange

Control organism/ material

Mycobacterium sp. Esch. coli Esch. coli Staph.aurues

Thin film blood smear Gram Iodine solution Spores

Esch. coli Staph.aureus

Formalin treated stool specimen with cysts

Bacillus species

ATCC No

25177 25922 25922 25923 25922 25923

Expected result

Pink red bacilli Blue bacilli Fluorescent bacilli/cocci Distinct staining of WBCs and RBCs Gram -ve bacilli Gram +ve cocci Visible cyst nuclei Spores stain one colour and bacillus stains with counterstain Quality control of stains need to be performed on weekly basis and also as and when a new lot of reagents for staining are procured

Staphylococcus aureus

, and shows Gram-positive cocci in grape like clusters. There are two major groups of Gram-positive cocci that are of medical importance: the

staphylococci

and the

streptococci .

When viewed microscopically, staphylococci appear in clumps, like bunches of grapes.

Staphule

is Greek for grapes. Streptococci form chains, and are named after

streptos

, the Greek word for twisted. These groups of bacteria can be distinguished because staphylococci produce an enzyme,

catalase

and streptococci do not. Catalase causes the conversion of hydrogen peroxide to water with the concomitant release of oxygen gas, seen as bubbles in the reaction tube. The catalase test is a more reliable test to differentiate staphylococci from streptococci than microscopic observation.

Streptococcus pyogenes

and shows Gram-positive cocci again, this time in chains.

Streptococci are classified according to their ability to break down blood in fresh blood agar plates.

Some streptococci have no effect on blood. These are the

non-haemolytic streptococci

(see below). The α

-haemolytic streptococci

cause partial breakdown of blood, and their colonies are surrounded by a greenish halo. The green pigment is thought to comprise the metabolic degradation products of haem. Because of the colour of halo that surrounds a haemolytic streptococci, they are often referred to as

"viridans" streptococci

(

viridis

is Latin for green). There is one α -haemolytic streptococcus that must be differentiated from the others. This is

Streptococcus pneumoniae

. This is the cause of pneumococcal pneumonia and meningitis, as well as less serious infections.

Streptococcus pneumoniae

is sensitive to optochin, an antimicrobial agent. All other viridans streptococci are resistant to optochin . Streptococcus Gram stain

Greening Streptococcus In Blood Agar

The

ß-haemolytic streptococci

cause the complete breakdown of blood in fresh blood agar plates. The colonies are surrounded by haloes that are completely clear. Clinically, the most important of the b-haemolytic streptococci is

Streptococcus pyogenes

. This belongs to the "Lancefield Group A" based upon its antigenic structure.

Streptococcus pyogenes

may be differentiated from other b-haemolytic streptococci on the basis of its sensitivity to the antibiotic bacitracin. The most important of the non-haemolytic streptococci are the

enterococci

such as

Enterococcus faecalis

and

Enterococcus faecium

. Until the early 1990's these bacteria were classified in the genus

Streptococcus

but molecular biological techniques have shown that they are sufficiently distant from other streptococci to warrant being placed in their own genus. As their names imply, these bacteria can be found in the gut, and can grow in the presence of bile salts.

• Beta-Hemolytic Streptoccus; group of bacteria, most common of which is

Streptococcus pyogenes

, which may cause Strep Throat, Scarlet Fever, Glomerulonephritis, or Rheumatic Fever. Hemolysis on blood agar. LM

Gram-positive Bacilli The genus Bacillus

The Gram-positive rods can be divided according to their ability or otherwise to produce spores.

Spores of Gram-positive rods are highly resistant structures that may add considerably to their pathogenic capacity. Sporing Gram-positive rods that are confined to the (somewhat confusingly named) genus

Bacillus

. Important members of this genus include

Bacillus anthracis

the cause of anthrax, and

Bacillus cereus

goddess of the harvest). a cause of food poisoning. (Ceres was a Roman The genus

Bacillus

also has members that produce clinically useful antibiotics, like

Bacillus polymyxa,

the source of polymyxin.

The image on the left is of

Clostridium perfringens

, stained from a clinical specimen using the Gram staining protocol. Spores can be seen as areas with no stain in a few of the dark fat rods of this bacterium. In culture, it is very difficult to persuade this bacterium to form spores but they are seen in clinical specimens. The spores are located towards the centre of the bacillus in this species and they do not cause the bacilliary body to swell.

In contrast, the spores of

Clostridium tetani

are located at the end of the cell and are wider than the bacilliary body. Sporing bacteria of

Clostridium tetani

have a characteristic 'drumstick' appearance, as seen in the image on the right. This is a spore stain made from a pure culture of these bacteria.

Clostridium tetani

Bacteria are gram-postive rods or bacilli with terminal spores that cause tetanus in humans, spore

Bacillus of Anthracis (Gram Stain)

Other Gram-positive bacilli

The motility of the

non-sporing Gram-positive rods

is an important attribute in distinguishing

coryneform bacteria

and

lactobacilli

from

listeria

.

Listeria monocytogenes

is an important human pathogen, and it is capable of a characteristic tumbling motility seen at 25 degrees C but not at 37 degrees C.

Lactobacilli

appear microscopically as long, slender rods that often grow in chains. They may appear "Gram-variable" with some parts of the cell appearing blue-black and other portions looking red. They tend to make their immediate environment too acid for other bacteria to tolerate. Some lactobacilli are important members of the vaginal commensal flora of women of child-bearing age. These are sometimes referred to as

Döderlein bacilli

. The lactobacilli are catalase-negative, and can thus be distinguished from the coryneform bacteria that do produce catalase.

The most infamous of the

coryneform

bacteria is

Corynebacterium diphtheriae

, toxigenic strains of which cause diphtheria. This gives the coryneform bacteria their alternative name -

diphtheroids

. They appear somewhat irregular in shape, and tend to cluster in Gram films. Some microbiologists think that this gives them the microscopic appearance of Chinese letters.

Propionibacteria

are coryneforms that cannot grow in the presence of air. A notable example is

Propionibacterium acnes

, associated with acne.

Gram stain of a

Corynebacterium

seen on a skin swab

Gram stain of Corynebacterium spp. demonstrating "Chinese letters" formations

Small, nonmotile, irregularly staining pleomorphic Gram-positive rods with club-shaped swelled ends but no spores; may be straight or slightly curved

C. diphtheriae

and related organisms are collectively termed coryneforms or diphtheroids Corynebacteria possess capsular (K) and somatic antigens (O) The top image is of a group of corynebacteria stained using Gram's method and clustered in their characteristic shape. Some bacteriologists consider these to resemble Chinese pictograms. An idealsied version is shown in the lower image

Pasteurella (Francisella) tularensis)

Bacteria, the gram-negative coccobacillus pathogen that causes Tularemia or Rabbit Fever. LM X500.

Description:

Gram Stain Smears of the Agents of Anthrax (

Bacillus anthracis

), Plague (

Yersinia pestis

), and Tularemia (

Francisella tularensis

), Demonstrating Comparative Morphology, Size, and Staining Characteristics ( Dennis et al., 2001 ). Tularemia, also known as rabbit fever or deer-fly fever, is a zoonotic disease caused by the gram-negative intracellular pathogen

Francisella tularensis

( Farlow et al., 2005

Francisella tularensis Growth at 72 Hours After Inoculation

These

Francisella tularensis

colonies show characteristic opalescence on cysteine heart agar with sheep blood (cultured at 37 C for 72 hours). Note: On cysteine heart agar, F tularensis colonies are characteristically opalescent and do not discolor the medium ( Dennis et al., 2001 ). •

F. tularensis

grows best in cysteine-enriched broth and thioglycollate broth. It grows best on cysteine heart blood agar, sheep blood agar, and on cysteine-supplemented agar such as buffered charcoal-yeast agar, Thayer-Martin agar, and chocolate agar. Selective agar may be useful when culturing materials from nonsterile sites, such as sputum.

Optimal Temperature:

37 C , ( Dennis et al., 2001 ).

Mycobacteria

The

mycobacteria

are a group of bacteria that are classified with other Gram-positive bacteria on the basis of their cellular architecture, but they possess a very waxy cell wall, and they rarely stain using conventional protocols such as the Gram stain. They require special staining techniques in order to be observed easily under the microscope. In the Ziehl Neelsen technique, a strong solution of carbol fuchsin is applied to the microscope slide which is then heated and held near boiling point for at least five minutes. This is to allow the stain to penetrate the mycobacterial cells. Following this treatment the stain will remain in the cell, even when the slide is treated with a mixture of inorganic acid and alcohol. To visualise any other material on the slide, methylene blue is applied as a counterstain. Because mycobacteria resist decolourisation with acids and alcohol they are sometimes called

acid alcohol-fast bacilli

. Important examples include

Mycobacterium tuberculosis

and

Mycobacterium leprae

. The former causes tuberculosis, and the latter is the cause of leprosy. It takes 6-8 weeks to grow

Mycobacterium tuberculosis

in artificial culture, and as yet

Mycobacterium leprae

cannot be grown in artificial culture.

Mycobacterium tuberculosis Bacteria in human sputum. LM X1600

Mycobacterium tuberculosis in sputum, stained by the Ziehl Neelsen technique

The red rods visible in the image above are cells of

Mycobacterium tuberculosis

seen in a film made from a sputum sample. They have retained the strong carbol fuchsin dye that has penetrated the cells following heating of the slide to steam heat, even though the film has been decolourised with a mixture of acid and alcohol. Because of this property, mycobacteria are referred to as acid alcohol-fast. The background material has been counterstained with methylene blue.

Gram-negative cocci

Medically, the most important of the Gram-negative cocci belong to the genus

Neisseria

.

Neisseria meningitidis

is an important cause of bacterial meningitis, and

Neisseria gonorrhoeae

causes gonorrhoea. Members of the genus

Neisseria

are most often seen in pairs, and are hence sometimes referred to as diplococci. They are very vulnerable to drying, and can only be cultivated in an atmosphere where the concentration of carbon dioxide is greater than that found in air. In the laboratory, carbon dioxide incubators are used that maintain a moist environment with a carbon dioxide concentration of 5-10%

Gram-negative cocci

Neisseria gonorrhoeae

in pus from a urethral discharge This bacterium is typically found in pairs, known as

diplococci

. Here the diplococcal cells are seen within a polymorphonuclear leukocyte seen in pus from a urethral discharge. This is sufficient information to make a provisional diagnosis of gonorrhoea, which will be confirmed following a positive culture result,

Gram-negative bacilli

Enterobacteriaceae This image shows the typical appearance of

Escherichia coli

when stained using the Gram staining method The

Enterobacteriaceae

are a large family of medically important Gram-negative bacilli. They can grow in the presence or absence of oxygen, and are frequently found in the guts of humans and other animals, and hence their name. They are differentiated from one another largely on the basis of their metabolic behaviour and on their antigenic structure. Some, like

Escherichia coli

and members of the genus

Klebsiella

can ferment lactose to produce acid, whereas others including

salmonellas, shigellas

and

proteeae

cannot and are thus known as

non-lactose fermenters (NLF's)

. There are almost 2,000 different varieties of salmonella recognised on the basis of differences in their surface antigens. Members of the genus

Proteus

are so highly motile that a single colony can grow to swarm over the entire surface of a Petri dish after overnight incubation. This is how the genus acquired its name, from Proteus, the Greek sea god who had the miraculous ability to change his shape. The family

Enterobacteriaceae

include

Yersinia pestis

, the cause of plague,

Salmonella typhi,

the cause of typhoid,

Shigella dysenteriae

, the cause of bacilliary dysentery, and

Salmonella enteritidis

implicated in many cases of food poisoning.

Mixed bacterial colonies on MacConkey agar, Escherichia coli (red) and Salmonella typhimurium (white).

Some bacteria are enclosed within a

capsule

.

This protects the bacterium, even within

phagocytes

, helping to prevent the cell from being killed.

Encapsulated bacteria grow as

'smooth

' colonies, whereas colonies of bacteria that have lost their capsules appear

rough

. Rough colonies do not generally cause disease. Encapsulated bacteria do not succumb to intracellular killing as easily as bacteria that lack capsules.

Cultures of Salmonella typhimurium on tryptone glucose agar (left) and on MacConkey agar (right).

Salmonella typhosa

: gram negative, brightfield. LM X500

Conventional Biochemical tubes for Shigella

spp

Conventional Biochemical tubes for Salmonella

spp

TABLE 1: BIOCHEMICAL IDENTIFICATION OF ENTERIC Pathogens K I A M I O LIA U NaCl Identification

S. typhi S. paratyphi A K K A AG + - + + - - - + K K K A - - - - Salmonella gallinarum K A V - - V K V - - Salmonella pullorum

Other

Salmonella

spp.

Shigella dysenteriae Shigella flexneri Shigella boydii Shigella sonnei Aeromonas

Spp.

Plesiomonas

spp.

Vibrio cholerae V. parahaemolyticus V. fluvialis K K K @ K K K K K K K K A AG A * A A A A A A A A V + - - - - - - - - - - - - - + - + + + + + - - V V V - V + + + V + + + - V + - - - + - K K K K K K K K K K K K K K A V K A A A A A - - - - - - - - - - - - - - - - - + + + + + - - - + - + + + + + +

Symbols: KIA: Kligler Iron Agar, MIO: Motility Indole Ornithine Agar, LIA: Lysine Iron Agar, K: alkaline, A: acid, G: gas, S: slant, B: butt, Ure: urease, Oxid: oxidase, * Some strains produce gas. @ Some strains ferment lactose (KIA=A/A) according to Farmer and Kelly, (1991)

+ + - -

Pseudomonads

Members of the family Enterobacteriaceae do not elaborate the enzyme complex known as "

oxidase

", whereas many Gram-negative bacteria do.

Pseudomonas aeruginosa

is an oxidase-positive Gram-negative bacillus that is an obligate aerobe. It cannot be grown in the absence of oxygen. It is responsible for wound infections, and the bacteria in this species produce a soluble pigment. Culture of Pseudomonas aeruginosa Bacteria on blood agar.

Curved Gram-negative rods

• • The

vibrios

and

campylobacters

are Gram-negative rods that appear curved or spiral in shape. These bacteria are commonly found in natural waters, both fresh-water and marine.

Vibrio cholerae

causes cholera, a waterborne infection.

Campylobacters

have only been recognised as human pathogens since the late 1970's, although they have been long considered to be animal pathogens. Campylobacters are now responsible for more cases of bacterial enteritis annually than salmonellas. The bacterium

Helicobacter pylori,

found associated with stomach ulcers, is a good example of a bacterium that has undergone a radical change in taxonomy. When they were first observed, they could not be cultured and were called "campylobacter-like organisms". Conditions were then discovered that allowed these bacteria to be grown in artificial culture, and they were first called

Campylobacter pyloridis

. This was then found to be linguistically incorrect, and the name was corrected to

Campylobacter pylori

. Later, molecular studies showed that it was not very closely related to the other campylobacters, and they then became the type species of a new genus,

Helicobacter

. This entire process of taxonomic change took less than five years.

Campylobacter Species

speciation chart Skirrow Campy plate primary plate 42 degrees C Microaerophilic Oxidase Test Positive Gram Stain Gram negative curved rods Hippurate Hydrolysis Oxidase Test negative Not

Campylobacter spp

Positive

C.jejuni

Negative Indoxyl Acetate Hydrolysis positive

C.coli

positive Catalase test negative

Campylobacter

species negativ

C.upsaliensis

• • •

Other Gram-negative bacilli

Some Gram-negative bacilli appear so short that they resemble cocci in the light microscope. Because of this they are sometimes called

cocco-bacilli

. These include members of the genus

Moraxella,

related to the neisserias, and also members of the genus

Acinetobacter

. Members of this genus are increasingly associated with hospital-acquired infection. Some Gram-negative bacteria are very

fastidious

(fussy) in their nutritional requirements. Members of the recently recognised genus

Legionella,

some of which cause atypical pneumonias like Legionnaires' disease, require higher levels of

iron

and

cysteine

than are usually present in bacteriological media, and they grow best in media that incorporate

activated charcoal

to adsorb their toxic metabolic products. Similarly bacteria of the genus inhibit their own artificial culture. These bacteria also grow best on media that contain activated charcoal.

Bordetella Bordetella pertussis,

also generate toxic metabolic products that the cause of whooping cough, is an important member of this genus. At one time, species of the genus

Bordetella

were classified in the genus

Haemophilus

, but they were re-classified. This is partly because they require neither the

X-

nor the

V-factor

for growth such as required by members of the genus

Haemophilus

. The

X-factor

and the

V-factor

is has now been identified as

nicotinamide adenine dinucleotide

or

NAD

.

haem

Haemophilus

,

influenzae

requires both X- and V-factors for growth whereas

Haemophilus parainfluenzae

requires just the V-factor to support its growth, since it can elaborate its own supply of haem. The most important group of

obligately anaerobic Gram-negative bacilli

are the

bacteroides

. This is a heterogeneous group that form part of the human commensal flora, and that are also implicated in anaerobic infections. The taxonomy of the anaerobic Gram-negative rods is currently undergoing radical revision.

X and V dependence in

Haemophilus influenzae

The pathogenic

Haemophilus influenzae

can be differentiated from its non-pathogenic relative,

Haemophilus parainfluenzae

, by its dependence on an external supply of two growth factors, known as 'X' (haem) and 'V' (NAD).

Haemophilus influenzae

requires both, while

Haemophilus parainfluenzae

can make its own haem. requires only the 'V' factor, since it In the image above, the bacteria can only grow around the disc containing both 'X' and 'V' factors, indicating that it is the pathogen,

Haemophilus influenzae

. X and V dependence may also be demonstrated by the phenomenon of 'satellitism'. The second image above illustrates this. A lawn of test bacteria is plated onto a fresh blood agar plate. This provides a supply of haem. An inoculum of

Staphylococcus aureus

, which can provide NAD, is placed on the plate and the culture is incubated. Haemophili can be seen growing larger near to the staphylococcal colony, where the supply of NAD is greatest. Colonies of haemophili growing further from the staphylococcal colony are proportionately smaller, as the supply of NAD from the staphylococcus diminishes. This test is good for detecting haemophili, but cannot distinguish the pathogenic

Haemophilus influenzae

from

Haemophilus parainfluenzae

.

•

Haemophilus influenzae

Haemophilus influenzae Bacteria, a gram-negative rod that causes Bacterial Meningitis. LM X500 .

(Haemophilus influenzae) and (Staphylococcus) satellite test (Haemophilus influenzae) and (Staphylococcus aureus) on blood agar, culture (Haemophilus influenzae) and (Staphylococcus) satellite test

Quality control procedures for commonly used tests Procedure/ Test Catalase Coagulase Indole Methyl red Oxidase ONPG disc Oxidase disc Voges Proskauer Bacitracin disc Optochin disc Control organism

Staph aureus Streptococcus species Staph aureus Staph epidermidis Esch coli Enterobacter aerogenes Esch coli Ent aerogenes P. aeruginosa Esch. coli Enterobacter aerogenes Esch. coli Streptococcus group A Enterobacter faecalis Strept. Pneumoniae Strept. viridans Esch. Coli Proteus vulgaris P aeruginosa Esch. coli

+ – + – + – + – + –

Expected result

+ – + – + – + – + –

Expected reaction

Bubbling reaction No bubbling Clot formation in 4 hours No clot Red ring at surface Yellow ring at surface Instant red colour No colour change Purple colour in 20 seconds No colour in 20 seconds Red colour No colour change Zone of inhibition No zone of inhibition Zone of inhibition No zone of inhibition Yellow colour No change in colour Purple colour in 30 seconds No change in colour

Quality control procedures

• • It is also essential to undertake quality control procedures at regular intervals. These should be performed: With each new batch of reagents • • With each new vial of reagent Daily for catalse, oxidase, and coagulase • Weekly for bacitracin, optochin and ONPG • A test procedure not giving anticipated results with the control organisms should not be used till such time that remedial steps have been taken to correct the

Basic sets of drugs for routine susceptibility tests Set 1 Set 2

Staphylococcus Intestinal

Enterobacteriaceae

Urinary Blood and tissues Pseudomonas aeruginosa

Benzylpenicillin Oxacillin Erythromycin Tetracycline Chloramphenicol Ampicillin Chloramphenicol Co-trimoxazole Nalidixic acid Tetracycline Sulfonamide rimethoprim Co-trimoxazole Ampicillin Nitrofurantoin Nalidixic acid Tetracycline Ampicillin Chloramphenicol Co-trimoxazole Tetracycline Cefalotin Gentamicin Piperacillin Gentamicin Tobramycin Gentamicin Amikacin Co-trimoxazole Clindamycin Norfloxacin Norfloxacin Chloramphenicol Gentamicin Cefuroxime Ceftriaxone Ciprofloxacin Piperacillin Amikacin Amikacin

Factors influencing zone size in antibiotic susceptibility testing Factor

Inoculum density Timing of disc application Temperature of incubation Incubation time Size of the plate Depth of the agar medium Proper spacing of the discs Potency of antibiotic discs Composition of medium Acidic pH of medium Alkaline pH of medium Incubation in the presence of CO 2 Addition of thymidine to medium Addition of defibrinated blood

Influence

Larger zones with light inoculum and vice versa If after application of disc, the plate is kept for longer time at room temperature, small zones may form Larger zones are seen with temperatures < 35 o C Ideal 16-18 hours; less time does not give reliable results Smaller plates accommodate less number of discs Thin media yield excessively large inhibition zones and vice versa Avoids overlapping of zones Deterioration in contents leads to reduced size Affects rate of growth, diffusion of antibiotics and activity of antibiotics Tetracycline, novobiocin, methicillin zones are larger Aminoglycosides, erythromycin zones are larger Increases zone size of tetracycline and methicillin Decreases activity of trimethoprim Decreases activity of sulfonamides On chocolate agar, decreased activity of Reading of zones Sulfonamides, trimethoprim, aminoglycosides Subjective errors in determining the clear edge Chelating agents such as cal-cium, magnesium and iron Decreases diffusion of tetracycline and gentamicin

Need for quality control in susceptibility test

• The final result of a disc diffusion test is influenced by a large number of variables. Some of the factors, such as the inoculum density and the incubation temperature, are easy to control, but a laboratory rarely knows the exact composition of a commercial medium or the batch-to batch variations in its quality, and it cannot take for granted the antimicrobial content of the discs. The results of the test must, therefore, be monitored constantly by a quality control programme which should be considered part of the procedure itself.

• The precision and accuracy of the test are controlled by the parallel use of a set of control strains, with known susceptibility to the antimicrobial agents. These quality control strains are tested using exactly the same procedure as for the test organisms. The zone sizes shown by the control organisms should fall within the range of diameters given in Table 11.4. When results regularly fall outside this range, they should be regarded as evidence that a technical error has been introduced into the test, or that the reagents are at fault. Each reagent and each step in the test should then be investigated until the cause of the error has been found and eliminated

Antibiotic

Amikacin Ampicillin Ceftriaxone Cephalothin Chloramphenicol Ciprofloxacin Clindamycin Erythromicin Gentamicin Nalidixic acid Nitrofurantoin Norfloxacin Oxacillin Penicillin G Piperacillin Tetracycline Tobramycin Trimethoprim sulfamethoxazole

Quality Control – Susceptibility of Control Strains* Disc potency

30 mg 10 mg 30 mg 30 mg 30 mg 5 mg 2 mg 15 mg 10 mg 30 mg 300 mg 10 mg 1 mg 10 units 100 mg 30 mg 10 mg 5 mg 1.25/23.75

18-22 17-28 18-24 26-37 – 19-28 19-29 19-26 24-32

Staph.aureus

(ATCC 25923)

20-26 27-35 22-28 29-37 19-26 22-30 24-30 22-30 19-27 –

Diameter of zone of inhibition (mm )

20-25 28-35 – – 24-30 18-25 18-26 21-28 24-32

Esch.coli

(ATCC 25922)

19-26 16-22 29-35 15-21 21-27 30-40 – – 19-26 22-28

P.aeruginosa

(ATCC 27853)

18-26 – 17-23 – – 25-33 – – – – – – 16-21 – 25-33 – 19-25 – –

Troubleshooting guide for disc diffusion test in antibiotic susceptibility testing Aberrant results

Tetracycline zone too small Aminoglycoside zone too small Aminoglycoside zone too large

Probable cause

pH of medium too low pH of medium too high Ca 2+ and/or Mg 2+ level too high in medium Ca 2+ and/or Mg 2+ level too low in medium Too large zone on control plates Zone universally too small on control plates Methicillin zone indeterminant in disc test Carbenicillin zone disappears with

Pseudomonas

control Single disc result above or below control limits Inoculum too light Nutritionally poor medium Slow growing organisms (not seen with controls) Improper medium depth (too thin) Inoculum too heavy Methicillin degraded by strong b lactamase producing staphylococci Resistant mutant has been selected for testing Colonies within zone of inhibition Zones overlap Zones indistinct Zone within zone phenomenon Error in reading, fuzzy zone edge, transcription error, bad disc Disc may not be pressed firmly onto agar surface Mixed culture Resistant mutants within zone Discs too close together Poorly streaked plates Swarming

Proteus species

Feather edge of zones around penicillin or ampicillin discs usually with b lactamase negative strains of

Staph.aureus

PCR

Gram negative cell wall Gram positive cell wall

Back to FRET

F

luorescence

R

esonance

E

nergy

T

ransfer

Reporter-Quencher probe system that allows the detection, and

-

quantifying, of nucleotide

amplification

in real time.

R Q

e-

R Q

__________________________________________________________ TaqMan

Bacteria

The bacterial cell

lacks a membrane-bound nucleus

. Because of this, bacteria are described as prokaryotes,

"pro-"

meaning "before" and

"-karyon"

from the Greek word for a "nucleus". There are three basic shapes that bacterial cells adopt. They are either round, rod shaped or spiral. Round bacteria are referred to as cocci (

singular

: coccus), and rod shaped bacteria are known as bacilli (

singular

: bacillus). The term 'bacillus' meaning a rod-shaped bacterium should NOT be confused with the genus of bacteria known as '

Bacillus

'.

Staphylococci Sporing cells of

Clostridium tetani

Note spores do not stain and in this case cause the bacilli to swell

Irregular cells of

Corynebacterium diphtheriae

Diplococcal cells of

Streptococcus pnuemoniae

Sporing cells of

Bacillus anthracis

Note spores do not cause the bacilli to swell in this species

Various shaped cells of

Haemophilus influenzae

Streptococci Spiral cells of

Treponema pallidum

This bacterium causes syphilis and is so slender that it cannot be seen using conventional light microscopy. It is most easily visualized using dark-ground microscopy.

Curved rods of Vibrio cholerae

• Strains of

Streptococcus pnuemoniae

capsules do not cause disease. All the bacteria that cause meningitis are encapsulated. that lack • Suspending bacteria in them. This is the

India ink

is an easy way of demonstrating capsules. Ink particles cannot penetrate the capsular material and encapsulated cells appear to have a halo around

Quellung

reaction. • In the 'Quellung' reaction, bacterial cells are resuspended in antiserum that carries antibodies raised against the capsule. This causes the capsule to swell, and this can be easily visualised by suspension in India Ink. The ink particles cannot penetrate the capsule, which this appears as a halo around the bacterial cells.

The Quellung reaction

Endospores (or simply spores)

• A few species of bacteria have the ability to produce highly resistant structures known as endospores (or simply spores). These resist a range of hazardous environments, and protect against heat, radiation, and desiccation. • Endospores form within (hence

endo-

) special vegetative cells known as

sporangia

(singular

sporangium

).

• • • • • • Diseases caused by sporing bacteria include

botulism

(

Clostridium botulinum

),

Ggas gangrene

(

Clostridium perfringens

),

Ttetanus

(

Clostridium tetani

)

Acute food poisoning

(

Clostridium perfringens

, again) All these bacteria are 'anaerobic'. The aerobic sporing bacteria can also cause disease.

Anthrax

Bacillus anthracis

.

Bacillus cereus

causes is caused by

two types of food poisoning

.

Microfungi

Microfungi

All fungi are eukaryotic. Most possess a cell wall made of chitin: a polymer of

N

-acetyl glucosamine that is found in the cell walls of the majority of fungi. It is also a major component of the exoskeleton of arthropods such as insects. The cell walls of plants are made of

cellulose

whereas the walls of fungal cells are made of

chitin

and other polymers. Chitin is also the material found in insect shells. of the genus

Cladosporium

. Who has not seen

Penicillium spp

. growing on stale bread?

These are all

moulds

: fungi that grow in mats of tiny filaments known as

hyphae

(singular: hypha, Greek for a thread) or

mycelia

(singular: mycelium, from the Greek mukes, meaning a mushroom). These may or may not be subdivided into separate compartments by cross walls known as

septa

(singular: septum).

Aseptate and septate mycelia

Moulds are multicellular organisms.

There are, however,

unicellular fungi

: the

yeasts.

Most familiar of the yeasts is

Saccharomyces cerevisiae

. Depending upon your viewpoint, this is baker's yeast, used in the production of leavened bread, or brewer's yeast, used in alcohol production.

Yeasts grow and multiply by budding daughter cells off from a mother cell.

Budding Yeasts

Fungi can multiply either sexually or asexually .

In classifying fungi, great weight is placed upon the appearance and structure of sexual fruiting bodies.

Any fungus that does not exhibit a recognizable sexual structure is difficult to classify. They are grouped in the collection known as "

fungi imperfecti

". Among the most important of fungi imperfecti are members of the genus

Penicillium.

It is from these fungi that we derive penicillins. These were the first true antibiotics and are still among the most used antimicrobial agents world-wide.

Fungi also produce a staggering variety of spores. These may be produced asexually or sexually and are important in the identification of moulds.

Examples of asexual spore structures produced by fungi

The umbrella is the spore-bearing structure once it has discharged its load Asexual spores can be enclosed within specialised sacs The umbrella is the spore-bearing structure once it has discharged its load

Examples of sexual spore structures produced by fungi

Spores within an enclosed structure - a

cleistothecium

The warty

zygospore

is suspended between two mycelia of different mating types Spores contained inside a more open structure - a

perithecium

Moulds cause a variety of common, superficial infections such as

ringworm

and

athlete's foot

. In compromised individuals they can cause much more severe infections but these are rare. The most common yeast infection is

"thrush"

caused by

Candida albicans

. Under certain conditions, some yeasts have the ability to develop

pseudomycelia

. This happens, for example, when the commensal form of active thrush.

Candida albicans

causes

"Pseudomycelia" produced by Candida albicans

Gram-stain of vaginal smear showing

Candida albicans

, epithelial cells, and many gram-negative rods

Gram-stain of vaginal smear showing Candida albicans epithelial cells and many gram-negative rods. (1,000X oil)

Viruses

Viruses

• • • • • Viruses are obligate intracellular parasites (requires to live within a cell in its host). They comprise a

nucleic acid core

wrapped in a

protein coat

. Some viruses have an

envelope,

made of lipid and usually derived from the cell in which they grow. Other viruses are

naked

and just have their protein coat exposed, protecting the nucleic acid within the centre of the structure. Specific viruses attack specific types of cells. Respiratory syncytial virus infects only the cells of the respiratory tract, for example.

There is a special class of virus that attacks bacteria. These are the

bacteriophage

.

Most viruses are simple in shape: round, rod-shaped, icosahedral, brick-shaped or bullet-shaped Viruses have a nucleic acid core, either

DNA

or

RNA

but not both. Retroviruses are unusual in that the virion carries an RNA copy of the genome but upon infection of a host cell a cDNA copy of the virus genome is made using the enzyme reverse transcriptase

Human immunodeficiency virus - the cause of AIDS

Around the nucleic acid core lies a

protein coat

, made up of units called

capsomeres

. Some viruses also have an

envelope

derived from the host cell membranes. The envelope may be either baggy or tight, depending upon the nature of the virus.

•.

Rabies virus Adenovirus - associated with respiratory and gastrointestinal disease Herpes simplex virus (note the lipid envelope) A T-even bacteriophage that can infect Escherichia coli