Simple Laboratory Methods in Microbiology

Proper Sampling in Clinical Microbiology Laboratory Collection of good quality specimens: Depends on The optimal time of specimen collection . The correct type of specimen Well collected specimens with minimum contamination from normal flora of the patient or the person collecting the specimen. Adequate amounts of each specimens and appropriate no. of specimens Clearly labeled safe specimens Optimal time of collection of collection of specimens: Specimens for the culture of bacteria collected before the start of antibiotic therapy Blood cultures and blood films for malarial parasites are best collected just as the patient’s temp. starts to starts to rise, however, when infective endocarditis is suspected , three blood culture sets collected with 24 hour irrespective of patient temp. Specimens for virus isolation are most likely to give positive results when collected during the most acute stages of the disease Serology is satisfactory when four fold or greater rising antibody titre is demonstrated in pained sera. The 1st serum sample as early as possible in the disease course. Second in the convalescent stage 2-Correct types of specimens Examples: Bacterial meningitis---------------blood cultures, CSF collacted Suspected gonorrhea------cervical, urethral and rectal swab should be collected rather than high regional subs. 3-Well collected specimens with minimum contamination from the normal flora: Poor quality specimens include saliva instead of sputum or a salivary – mucoid sputum sample instead of a muco purulent sputum. Mid stream urine reed careful collection to a void excess contamination by genital flora. A throat swab should not touch the buccal mucosa and the tongue depressed by a spatula. Vaginal speculum should not be wet with antiseptic solution during collection of high vaginal swabs with care not to touch the lower region or perineum Strict septic and antiseptic techniques are used for blood and CSF cultures to avoid contamination from Skin flora or from the doctor. Adequate amounts and Appropriate number of specimens The volume of blood for culture from adult 5-10 ml per bottle and in children and neonates 1-5ml per bottle. Collection of early morning sputum specimens, and collection of adequate amount of early morning urine specimen for 3 successive days is required for the isolation of M.T.B. Patients with diarrhea at least 2 specimens of faces is collected for culture of Salmonellae or Shigella. Serological investigations usually require paired sera. Clearly labeled and safe specimens Specimens for microbiological investigations should be placed in leak – proof containers, and each container should be enclosed in plastic bag. The hazards to staff handling leaking container s include acquiring enteric infection from feces, T.B. from sputum of an open case of pulmonary. T.B. and viruses s such as HCV , HBV, HIV, from leaking blood. Transport of specimens to the laboratory Many pathogenic organisms don’t survive for long in clinical specimens kept at room temp. Examples include gonococci, Haemophilus, Bacteroides, or aerobic cocci and most viruses. On the other hand, some organisms contaminating specimens from the normal flora such as coliform and coagulase –ve staphylo cocci, may rapidly grow in specimen Kept at room temp. -Urine or sputum specimens should reach the laboratory within 2h.of collection when even possible. If delay are expected immediately inoculated into transport media . Transport of specimens to the laboratory Many pathogenic organisms don’t survive for long in clinical specimens kept at room temp. Examples include gonococci, Haemophilus, Bacteroides, or aerobic cocci and most viruses. On the other hand, some organisms contaminating specimens from the normal flora such as coliform and coagulase –ve staphylo cocci, may rapidly grow in specimen Kept at room temp. -Urine or sputum specimens should reach the laboratory within 2h.of collection when even possible. If delay are expected immediately inoculated into transport media . Laboratory procedures for microbiological diagnosis Laboratory procedures for microbiological diagnosis Include the following steps: -Naked eye examination of Specimens -Microscopy. -Detection of microbial antigens. -Isolation of microbes - Antibiotic sensitivity -Serology. -Molecular biology techniques. -Gas – liquid chromatographic techniques. -Skin tests. I . Naked eye examination of specimens:- This helps to determine whether a specimen is suitable or no. A saliva sample instead of an expectorated sputum sample should be discarded. Turbid CSF, is an immediate evidence of infection A foul – smelling pus specimen may suggest presence of anaerobes. A rice water stool sample may indicate vibrio cholera infection Anchory sauce sputum sample would suggest invasive amoebiasis in lungs. -Sulpur granules in pus would indicate actinomycosis II. Microscopy: 1-Wet preparation for light microscopy in examination of CSF, urine , body fluid for evidence of pus cells, and organisms. Vaginal secretion trichomonas and Candida Skin, nail , hair (in KaoH) -evidence of fungus Dark ground illumination to look for spirochates, treponema pallidum in suspected 1ry or 2ry . II. Microscopy: 1-Wet preparation for light microscopy in examination of CSF, urine , body fluid for evidence of pus cells, and organisms. Vaginal secretion trichomonas and Candida Skin, nail , hair (in KaoH) -evidence of fungus Dark ground illumination to look for spirochates, treponema pallidum in suspected 1ry or 2ry . Gram stained smear: It may help of saving lif -Important in rapid diagnosis of bacterial meningitis on exam of CSF deposit. Diagnosis of strepto pneumonia in sputum smear Identification of colonies appearing on culture media Gram – stained smear may give + ve results al though the subsequent cultures are – ve as a result of given antibiotics In vincent angina: stained smear the only means of diagnosis Acid fast stain of sputum allow rapid diagnosis of open plm. T.B demonstrating acid fast bacilli. In other, clinical specimens such as urine , peritoneal fluid, CSF In vincent angina: stained smear the only means of diagnosis Acid fast stain of sputum allow rapid diagnosis of open plm. T.B demonstrating acid fast bacImmunoflourescent microscopy is important for rapid diagnosis of viral infection e.g. RSV in infants and children Herpes simplex CMV in urine throat swab. Rabies in brain biopsy specimen Clamydia trachomatis in conjunctiva scraping Also in serological antibody test e.g. -Fluorescent treponema antibody -fluorescent amoebic antibody. illi. In other, clinical specimens such as urine , peritoneal fluid, CSF III- Detection of microbial antigens: - Immunoelectrophoresis . e.g Pneumococcal polysaccharide Ag. may be detected in sputum, serum , urine of patients by immunoelecto phoresis, when patients have already given antibiotics where as conventional sputum blood cultures are negative -HbsAg. Detected using ELISA or latex test -Cryptococcal antigen in CSF in patients with cryptoccal meningitis. -Rota virus Ag. Using Elisa in feces of diarrheic children infected with Rota v. -Chlamydia trachomatis Ag using Elisa in conjunctiva scrapping in patients with active trachoma. -Detection of meningococcal, hemophiluss, preumococcal Ag. In CSF specimens by latex -Commercially available monoclonal specific antibodies for detection of Ag. In clinical specimens or cultures including various streptococcal, staphylococcal species, Neisseria, Candida spp. Chlamydia trachomatis, and Rotaviruses, CMV, herpes, Adeno viruses, RSV, influenza viruses. IV- Isolation of microbes Is the most reliable way in which a diagnosis can be confirmed and for obtaining antimicrobial susceptibility results. -Isolation of bacteria or fungus from specimens such as Blood, CSF (which are normally sterile) are easy to interpret. -Bacterial or fungal isolation from specimens collected from sites with normal flora are often difficult to interpret. -Choice of media is important according to type of specimen and suspected organism. Virological or Chlamydia isolation methods. Need preparation of the cell line required for support growth of suspected infecting virus. VI-Molecular biology techniques: Increasingly important for rapid diagnosis of infections for epidemiological investigations and for monitoring antimicrobial therapy Also, important for research on the pathogenesis of infection, the developmental new raciness and immune therapeutic agents. VII_Gas Liquid chromatographic techniques: Become increasingly useful for the rapid detection of anaerobic infections Specimens of pus from abdominal, gynecological or brain abscesses may be shown to have multiple volatile fatty acids present which indicate anaerobic infection with a few hours of collection of the specimen of pus and this may affect decisions about the chemotherapy of infection VIII- Skin test: Of limited value for diagnosis of infection ex: Mantaux skin test Histoplasmin test Casoni test Schick test . XI-Antimicrobial susceptibility testing Culture of Normal Flora Organisms MATERIALS: • Sterile cotton swab • Bacteriological loop • Nutrient Agar plates • Biohazard discard container DIRECTIONS: 1. Carefully swab the selected skin surface site in an area approximately 4 cm in diameter. Swab thoroughly, rotating the swab to ensure that a good inoculum has been obtained. 2. Transfer the bacteria to the agar culture dish by touching the swab to the agar surface in a single spot. Once you feel confident that a good inoculum has been transferred, the swab can be discarded in the biohazard container. 3. With the inoculating loop, proceed to streak in a second and third streak pattern (see illustration). Briefly, isolate bacterial colonies by pulling several streaks out from the original swab inoculum site. Use the method shown above to avoid tearing the agar. 4. Incubate the plate at 37oC for 24-48 hours. 5. Test the colonies that have grown by Gram staining; identify Staphylococcus aureus colonies by testing Gram-positive cocci using a specific latex agglutination reagent that binds to Staphylococcus aureus. Pure Culture Techniques Microbiologists have developed special techniques and equipment to isolate and grow pure cultures of microorganisms that are free from contaminating forms. This exercise is designed to give the student an introduction to these special techniques. It is important that techniques be practiced until some degree of skill is developed. Pure cultures are essential when identifying unknowns. During the course of the semester, each student will be expected to develop independently a patient-oriented case to correlate with each of 4 separate bacterial isolates. It is important, therefore, to maintain unique isolates in pure culture to be submitted with each case summary. Further instruction will be given during later labs. THE STREAK PLATE TECHNIQUE The procedure of streaking a plate with an inoculating loop is used to spread millions of cells over the surface of a solid medium so that some individual cells are deposited at a distance from all others. These cells grow and reproduce, forming an isolated colony. One or more colonies will be well separated from all others and represent a source of a pure culture. The procedure is similar to the one used for streaking from the collection swab. MATERIALS: • Streak plates from the previous lab session • Nutrient agar plates (NA) • Bunsen burner • Bacteriological loop DIRECTIONS: Examine the streak plates prepared during the previous lab period and locate a number of well-isolated colonies. Now you must transfer a portion of each colony to a separate agar slant. To "pick" a colony you will be using an inoculating loop. Sterilize the loop in the burner flame, let cool 3-5 seconds then touch the end of the loop to the isolated colony, picking up the microorganisms from the colony. Now re-cover the streak plate and pick up one NA plate. You will now be holding the inoculating loop in your right hand and the fresh NA plate in your left (lefties reversed). Remove the lid from the plate, place the inoculating loop at one edge of the plate and with a sweeping stroke, inoculate the agar using the same tri-streak method as used for the initial isolation. Replace the lid. Flame the loop and proceed to inoculate another plate from different colonies. Try to use colonies that are visibly different in morphology. Incubate the plates in the 37oC incubator until the next class period. There is nothing difficult about picking colonies and inoculating slants, but you must avoid contamination.

Identification of the Enterobacteriacae

THE ENTERIC BACILLI

Diseases: Enteric bacteriosis (hospital-acquired or nosocomial infections with various Gram-negative rods acquired by patients during hospitalization).

Etiologic Agents: Enterobacter, Escherichia, Klebsiella, Proteus, and Serratia.

Source: environmental sources, clinical sources, and normal flora

Pathogenesis: Bacteremia, pneumonia, urinary tract infections, wound infections, CNS infections, abscess formation in various organs, and colonization and infection of implants, prostheses, and catheters.

Laboratory Diagnosis: Bacteriologic isolation of etiologic agents from blood, other body fluids, wounds, and exudates. Antimicrobial susceptibilities tests characteristically reveal resistance to commonly used antibiotics. Molecular epidemiological analysis may identify resistance plasmids. Several companies manufacture diagnostic kits based on biochemical test properties of the organisms. The Enterotube method will be demonstrated.

THE LACTOSE FERMENTERS

E. coli INFECTIONS

Diseases: Intestinal infections (diarrhea, dysentery, hemorrhagic colitis). Urinary tract infections (asymptomatic bacteriuria, cystitis, pyelonephritis, prostatitis). Bacteremia. Neonatal meningitis.

Etiologic Agents: Intestinal infections - enterotoxigenic Escherichia coli (ETEC), enteroinvasive E. coli (EIEC), enteropathogenic E. coli (EPEC), and enterohemorrhagic E. coli (EHEC). Urinary tract infections - E. coli, usually strains carrying uropathogenic determinants. Bacteremia - E. coli, usually strains belonging to the same serogroups associated with urinary tract infections. Neonatal meningitis - E. coli (usually strains with K1 Ag).

Source: Intestinal infections - human intestinal tract, cattle (EHEC). UTI, Bacteremia and Neonatal meningitis- human intestinal tract

Pathogenesis: Intestinal infections - interaction of the intestinal mucosa with colonization factors, heat-labile (LT) and heat-stable (ST) enterotoxins (ETEC); enterocyte invasiveness factors (EIEC); enteroadhesiveness factors. Urinary tract infections - interaction of urinary tract mucosa with adhesins (type 1 fimbriae, P-fimbriae, X-adhesins); possibly hemolysin, O polysaccharides; immature host immunity; shock associated with endotoxin. Neonatal meningitis-acidic polysaccharide capsule K1.

Laboratory Diagnosis: Isolation of E. coli from the appropriate clinical samples. Kits are available commercially. These are based on the biochemical tests that will be presented in this lab section.

Adapted from Laboratory Diagnosis of Infectious Diseases, ed. A. Balows

A SIMPLIFIED BRANCHING FLOW DIAGRAM FOR GRAM-NEGATIVE RODS

ISOLATION OF ENTEROBACTERIACEAE

There are numerous plating media in use today, some selective, and others inhibitory. Most laboratories prefer to employ one selective medium such as SS agar, and one inhibitory medium, such as EMB or MacConkey agar for fecal specimens. For specimens other than feces or rectal swabs, a combination of MacConkey or EMB agar together with a BAP plate is usually sufficient.

Characteristics and Tests Used for the Species Identification of Enterics:

Identification of the Enterobacteriaceae is based on colony morphology on primary isolation media and on biochemical reactions. The following set of tests is a subset of those tests widely used in clinical laboratories:

·                     Utilization of carbohydrates (fermentation)

·                     The IMVC reactions:
I = production of Indole from tryptophan
M = Methyl Red Reaction
V = Voges-Proskauer test (Production of acetyl-methyl carbinol)
C = Utilization of Citrate

·                     Motility

·                     Production of Urease

·                     Production of H₂S gas

Carbohydrate fermentation pattern for an isolate can be determined using any of several commercially-available minaturized test configurations (Enterotube, API test strip). In the clinical lab these tests are usually performed using a Vitek automated analyzer and liquid culture isolates.

Automated Analysis System for Enteric Microorganisms

Hospital microbiology laboratories typically employ fully automated computerized methods for the identification and classification of enteric bacterial species. One example of such a system is the Automicrobic System (bioMerieux Vitek, Inc. Microscan) The system uses disposable polystyrene cards that include 30 reaction micro-wells. Each microwell contains biochemical substrates or dehydrated antibiotics. A filling module automatically fills each microwell with the test bacterium and seals the card. An incubation and reading module optically scans the cards on a 1-hour rotational basis as they are incubated. A computer module records the biochemical reactions and performs identification analyses. The final identification report can be available with as little as 6 hours of incubation. The total time required to culture and identify a suspect enteric pathogen, however, includes the initial primary culture for isolation, generally on MacConkey agar.

I) Primary isolation medium (Inhibitory).

Important Purpose and Differential Medium Ingredients

MacConkey agar

MAC Agar Formula
Bile salt (1.5g/l) & Crystal violet
Purpose: Recover the Enterobacteriaceae & related Lactose-fermenting Gram-negative enteric bacilli.
Inhibitor: Bile salt & Crystal Violet. Inhibit the growth of Gram-positive bacteria and some fastidious Gram-negative bacteria.
Carbohydrate Source: Lactose
Neutral Red, pH indicator

Eosin-Methylene-Blue agar (EMB)
Eosin Y and Methylene-blue

Purpose: Isolation & detection of the Enterobacteriaceae or Lactose-related coliform bacilli from specimens with mixed bacteria.
Inhibitor: Eosin & methylene blue. Inhibits the growth of Gram-positive bacteria and some fastidious G- organisms
Carbohydrate Source: Lactose
pH Indicator: Eosin & methylene blue combine to form a precipitate at acidic pH.

 Differential and Selective Culture of Bacteria

The Selective Media allow one type of organism to grow preferentially because of the components included in the agar. Examples are the agars that selectively inhibit the growth of most organisms, while allowing skin bacteria to grow (Mannitol-Salt Agar for Staph. culture), those that inhibit Gram-positive organisms due to bile salts (MacConkey agar) and dyes like basic fuchsin, eosin, and methylene blue (EMB agar).

The Differential Media allow different groups of bacteria to be distinguished based on their appearance and/or biochemical reactions. 

Blood agar as described for the culture of Streptococci, is one such agar. It allows bacteria to be distinguished based on hemolysis of the blood in the agar. However, it does not prohibit the growth of other types of bacteria, so it is not a selective media. Some strains of Staphylococcus produce beta-hemolysis on BAP.

Some differential media are sophisticated enough to allow preliminary identification of bacterial species to be made simply from culture results. MacConkey agar is both differential and selective. Since it contains lactose and neutral red dye, so lactose-fermenting colonies appear pink-to-red in color and are easy to distinguish from nonfermenting colonies. This is because of a reaction between the acid metabolic by-products of bacterial growth and the agar components.