Bacterial Culture: Different Types of Culture Methods

Bacterial culture is employed:

1. To isolate bacteria in pure culture.

2. To demonstrate their properties.

3. To obtain sufficient growth for antigen preparation.

4. For typing of microorganisms.

5. To determine antibiotic sensitivity.

 

METHODS OF BACTERIAL CULTURE:

STREAK CULTURE

• The medium in a petridish may be inoculated by loop, swab or other suitable device. Once the primary inoculum is made, a loop or straight wire can be used for spreading the material into four quadrants of the plate.

• A platinum or nichrome wire loop of 2-4 mm diameter with 2 to 3 inches long wire is first sterilized in the Bunsen flame and cooled by touching an uninoculated part of the medium. Then a loopful of specimen is gently smeared onto the surface of a well dried plate of medium near the peripheral area.

• The inoculum is then thinly spread in parallel lines in different segments of the plate. The loop is sterilized between different sets of streaks.

• The plate is incubated at 37°C for overnight. Confluent growth occurs at the primary site of inoculation and well separated colonies appear on the final series of streaks.

STROKE CULTURE

• It is done on agar slope for providing a pure growth of bacteria for slide agglutination and other diagnostic tests.

 

STAB CULTURE

• Stab culture is performed by a 4 inch long thick straight wire, charged with culture material, deep inside the agar.

• This technique is employed to demonstrate gelatine liquefaction, motility, oxygen requirement and maintenance of stock culture.

LAWN/CARPET CULTURE

• This technique is employed in antibiotic sensitivity testing (disc diffusion method) and in bacteriophage typing.

• Specimen of liquid culture or suspension of bacterial emulsion is poured on the surface of a culture plate and kept for a minute and then excess material is thrown out.

• Alternatively, the surface of the plate may be inoculated by a sterile swab soaked in liquid bacterial culture or emulsion.

POUR PLATE CULTURE

• Several tubes containing 15 ml agar medium in each are melted and left to cool in water bath at a temperature of 45-50°C. The inoculum to be tested is diluted in serial dilutions.

• One ml volume of each diluted inoculum is added to each tube of molten agar and mixed well. The content of each tube is poured into separate petridish and allowed to solidify.

• After overnight incubation the plates are examined for bacterial colonies. Colonies will be seen throughout the depth of the medium which are counted by colony counter.

• This technique is employed to estimate viable bacterial count in a suspension. It is also the recommended method for bacterial count in urine cultures.

SWEEP PLATE METHOD

• It is useful in studying microorganisms of a fabric e.g. a table cloth etc.

• The edges of the petri dish containing culture medium are rubbed over the cloth or fabric with the medium facing it.

• The medium is incubated. The dust particles of the cloth settle on the medium forming colonies which can be counted and estimated.

ADVANTAGES OF SOLID CULTURE MEDIA

• There is separate colony formation.

• By studying colonial morphology, presumptive identification of most bacterial species can be made.

• Quantitative bacterial count and relative proportion of different bacterial species can be made.

• Isolation of bacteria in pure culture can be made by picking isolated bacterial colonies and subculturing into fresh medium. This is necessary for full identification of bacteria.

LIQUID CULTURE MEDIA

• These are distributed in test tubes with cotton wool stoppers and screw-capped bottles or flasks.

ADVANTAGES OF LIQUID CULTURE MEDIA

• When bacteria are present in small numbers in the inocula, they will grow only in liquid media e.g. blood culture.

• Specimens containing inhibitory substances like antibiotics and other antibacterial substances get diluted by inoculation into the larger volume of the fluid medium.

• Liquid media are widely used for biochemical tests.

• These are also useful enrichment media like selenite F broth.

• Large inocula can be tested in liquid media, e.g. gauge for sterility test.

• Presumptive bacterial count in water sample is made in liquid media.

DISADVANTAGES OF LIQUID CULTURE MEDIA

• Isolation of bacteria in pure culture is not possible.

• Identification of bacteria is not possible.

INCUBATION OF BACTERIAL CULTURE

AEROBIC BACTERIAL CULTURE

• Most of the pathogenic organisms grow best at 37°C or body heat. The culture media, after inoculation, is incubated at 37°C in an incubator.

• Some bacteria require special temperatures for growth, eg. 45°C for campylobacters and 30°C for leptospires. Extra CO2 is needed for optimal growth of some bacteria, eg. B.abortus.

ANAEROBIC BACTERIAL CULTURE

• Anaerobic bacteria require incubation without oxygen.

• However a few anaerobes are aerotolerant eg. Clostridium histolyticum which may produce some growth on the surface of aerobic plates, while vast majority are strict anaerobes eg. Clostridium tetani.

• Anaerobiosis can be established by various methods.

Various methods of anaerobiosis:

• Displacement of oxygen

• Cultivation in vacuum in a vacuum dessicator

• Displacement of oxygen
• By an inert gas like hydrogen or nitrogen 
• By the use of Candle jar

• Absorption of oxygen by chemicals
• Pyrogallic acid
• Mixture of powdered chromium and sulphuric acid
• Gas-pak

• By displacement and combustion of oxygen
• McIntosh and Filde’s anaerobic jar

• By biological methods
• Incubating aerobic and anaerobic organisms together

• By incorporating reducing agents in media
• Thioglycollate broth
• Robertson’s cooked meat medium

Nasopharyngeal Swab and Aspirate-Collection and Transport

COMMON PATHOGENS FOUND IN NASOPHARYNGEAL SWAB

BACTERIA	GRAM POSITIVE: S. pneumonia, C. diphtheria, S. aureus, M. Ieprae
	GRAM NEGATIVE: H. inflenzae, N. meningitides, B. pertussis, B. Parapertusis, Klebsiella species.
VIRUSES	Respiratory viruses, Entero viruses

 

COLLECTION OF PRE-NASAL SWABS-NASOPHARYNGEAL SWAB

Infection with B. pertussis and B. parapertussis (Whooping cough) usually occurs in very young children who have not been immunized against the disease. The organisms are best isolated from pre-nasal swabs.

• Using a sterile cotton or alginate wool swab attached to an easily bent piece of wire, gently pass the swab along the floor of one nostril directing the swab downwards and backwards as far as the nasopharynx. 

• Taking care not to contaminate the nasal swab, replace it in its sterile container. Label and deliver immediately to the laboratory with a request form (B.pertussis does not survive on a swab). 

• If there is delay in transport, then insert the swab in a bijou bottle containing special bordetella transport medium.

 

COLLECTION OF ANTERIOR NASAL SWABS

• Mostly anterior nasal swabs are collected to detect carriers of pathogens like S. aureus, S. pyogenes, N. meningitidis, H. influenzae. 

• 1. Using a sterile cotton wool swab moistened with sterile peptone water, gently swab the inside surface of the nose. 

• 2. Put it in its sterile container, label & transport within 2 hours to the laboratory. 

• 3. If there is delay in transport then transfer it in Amies transportmedium.

 

COLLECTION OF NASOPHARYNGEAL ASPIRATES

• When it is not possible to obtain sputum from children with suspected pneumonia or bronchopneumonia, pathogens can often be isolated from a specimen of mucopus aspirated from the nasopharynx. 

• Gently pass a sterile catheter through one nostril as far as the nasopharynx. Attach a sterile syringe to the catheter and aspirate a specimen of mucopus. Dispense the specimen into a small sterile container. Label and deliver with a request form to the laboratory as early as possible. 

• lf there is delay in transport then make a swab of the aspirate and transfer in Amies transport medium.

Throat Swab: Collection and Transport

COMMON PATHOGENS FOUND IN THROAT SWAB:

BACTERIA	GRAM POSITIVE: Streptococcus pyogenes, Corynebacterium diphtheria, Corynebacterium ulcerans
	GRAM NEGATIVE: Vincent’s organisms (Borrelia vincenti and gram negative anaerobic fusiform bacilli)
VIRUSES	Respiratory viruses, Enteroviruses, Herpes simplex virus type 1
FUNGI	Candida albicans and other yeasts

 

NOTES ON PATHOGENS FOUND IN THROAT SWAB

• Steptococcus pyogenes is the commonest cause of streptococcal pharyngitis (sore throat) especially in young children & is also associated with rheumatic heart disease. 

• Corynebacterium diphtheriae cause a serious disease, diphtheria, producing a powerful and often fatal exotoxin. 

• C. albicans infection of the mouth (oral thrush) is often found with HIV disease. It also affects the patients who have been treated with antibiotics over a long period and patient with diabetes. 

• Infection with Vincent’s organisms causes Vincent’s angina, an ulcerative tonsillitis with tissue necrosis.

 

COLLECTION AND TRANSPORT OF 

THROAT SWAB

In a hospital with a microbiology laboratory:

For 8 hours before swabbing, the patient must not be treated with antibiotics or antiseptic mouth—washes.

1. In a good light and using the handle of a spoon to depress the tongue, examine the inside of the mouth. Look for - inflammation (Presence of any membrane, exudates or pus).

In diphtheria - greyish—yellow membrane

In streptococcal sore throat - tonsils are inflamed & covered with yellow spots.

In infectious mononucleosis — tonsils may be covered with white exudates.

In C. albicans inflection - Patches of white exudates.

2. Swab the affected area using a sterile cotton or alginate wool swab. Taking care not to contaminate the swab with saliva, return it to its sterile container.

3. Within 2 hours of collection, deliver the swab with a request form to the laboratory.

 

In a health centre for dispatch to a microbiology laboratory:

If there is delay in transport of throat swab, then transfer the swab in tubes containing 3-5 gm of desiccated silica gel (Here, C. diphtheriae, S. pyogenes & S. aureus remain viable for at least 3 days).

Blood culture: Collection and Transport of Body Fluids

Collection and Transport of Blood and Body

  Fluids for Culture

 

GENERAL CONSIDERATIONS:

The proper collection of sample is the most important step in the ultimate confirmation that a microorganism is responsible for the infectious disease process.

Failure to isolate the causative organism in an infectious process is not necessarily the fault of inadequate cultural methods; it frequently results from faulty collecting or transport techniques.

So, following are the general considerations recommended regarding the collection and transport of material for culture

• Whenever possible specimen should be collected before antimicrobial agents have been administered.

• The material should be collected where the suspected organisms is most likely to be found, with as little external contamination is possible.

• Another factor that contributes in successful isolation of organism is the stage of the disease at which the specimen is collected for culture.

• Specimens should be of a sufficient quantity to permit complete examination.

• The specimen should be placed in a sterile leak-proof container to prevent hazard to the handling staff.

• Provision should be made for the prompt delivery of specimen to the laboratory.

• One important thing is that clinicians should give sufficient clinical information to guide the microbiologist in selection of suitable media and appropriate technique.

• The clinicians must appreciate the limitations and potentials of the bacteriology laboratory and realize that a negative result does not necessarily invalidate the diagnosis.

• Laboratory personnel should reject specimens not obtained in a proper manner.

• Each specimen must be clearly labelled with the date and time of collection, patient’s name, number, ward or health centre.

• Each specimen must be accompanied by a request form which includes the following:

• Patient’s name, age, number and ward or health centre with name of consulting doctor.
• Type of specimen and date and time of its collection.
• Clinical diagnosis with patient’s history.
• Patient`s immune status.
• Any antimicrobial treatment that may be started at home or in the hospital.
• Specimens containing dangerous pathogens should be labelled as HIGH RISK and immediately after collection they should be sealed inside plastic bag or in a container with a tight — fitting lid.

• HIGH RISK specimens include:
• Sputum likely to contain M. tuberculosis.
• Faecal specimen that may contain V.cholera or S. typhi.
• Specimens from patients with highly contagious infections like HIV, Hepatitis, Viral haemorrhagic fever or plague.

 

BLOOD CULTURE

Blood culture is the most useful tool to detect

microorganism from cases of pyrexia of unknown

 origin.

 

DEFINITIONS:

• Bacteraemia: The presence of bacteria in blood is called bacteraemia. It is usually pathological although transitory asymptomatic bacteraemia can occur during the course of many infections and following surgical procedures. Bacteraemia occurs in diseases such as typhoid fever, brucellosis, leptospirosis and endocarditis.

• Septicaemia: The term septicaemia refers to a severe and often fatal infection of the blood in which bacteria multiply and release toxins into the blood stream. The commonest portals of entry for bacteraemia/septicaemia are the genitourinary tract, respiratory tract, abscesses, surgical wound infections, biliary tract and miscellaneous sites.

 

COMMON PATHOGENS CAUSING SEPTICEMIA

 

BACTERIA	GRAM POSITIVE: Staphylococcus aureus, Viridans streptococci, Streptococcus pneumonia, Streptococcus pyogenes, Enterococcus faecalis, Clostridium perfringens, Anaerobic streptococci.
	GRAM NEGATIVE: Salmonella typhi, other Salmonella species, Brucella species, Haemophilus influenza, Pseudomonas aeruginosa, Klebsiella species, Escherichia coli, Proteus species, Bacteroides fragilis, Neisseria meningitides, Yersinia pestis
OTHER BACTERIA	Mycobacterium tuberculosis, Leptospira species, Borrelia species, Rickettsiae and Bartonella bacilliformis
FUNGI	Candida albicans and other yeasts and occasionally Histoplasma capsulatum and other fungi that cause systemic mycoses

 

INDICATIONS FOR BLOOD CULTURE

• Septicaemia, an often life — threatening microbial invasion from an infected focus accompanied by increase in temperature, increase in pulse rate or chills followed by fever.

• Bacteraemia, which accompanies chronic infections such as disseminated gonococcal disease or severe infections exemplified by meningitis, pneumonia, or deep seated abscesses.

• Intravascular infections such as endocarditis, thrombosed blood vessels or those due to intravenous catheters.

• Bacteraemia of multisystem infections such as enteric fever, leptospirosis, brucellosis.

• Bacteremia secondary to traumatic insults and instrumentation such as puncture wounds, urinary tract catheterization, contaminated intravenous medication.

• Patients having fever of unknown origin (FUO).

 

TIMINGS FOR COLLECTION OF BLOOD FOR CULTURE

• In conditions like undrained abscesses, instrumentation of contaminated mucosal surfaces, manipulation of infected tissues, bacteria are transiently present in the blood stream.

• During early stages of typhoid fever, brucellosis, bacteria are continuously present in the blood stream.

• While in bacterial endocarditis, septic shock, organisms are released into the blood stream at fairly constant rate making timing of cultures unimportant.

• So blood should be collected at the time of the patient’s temperature beginning to rise. (Except bacterial endocarditis where blood can be collected at any time).

 

MEDIA FOR BLOOD CULTURE

• Blood culture media should contain a nutrient broth and an anticoagulant.
• (i) Tryptic soya broth.
• (ii) Brain heart infusion broth.
• (iii) Thioghycollate broth.

 

COLLECTION OF BLOOD

• Blood is collected by proper phlebotomy. Skin should be cleaned properly before collecting blood, this is necessary to reduce risk of introducing contaminants into blood culture media. 

• It is less desirable to draw blood through a vascular shunt or catheter since these prosthetic devices are difficult to decontaminate completely.

• It is also recommended to draw blood below an existing intravenous line if possible, since, blood above the line will be diluted with the fluid being infused.

• If the blood is not being inoculated directly into broth media, it must be transported with an anticoagulant sodium polyanethol sulfonate (SPS, Liquoid). 

• SPS is also anticomplementary, antiphagocytic and interferes with the activity of some antimicrobial agents, notably aminoglycosides. Heparin, EDTA and Citrate have been found to be inhibitory to a number of organisms.

 

Method:

Total aseptic precautions, just like minor surgical preparations are required.

(i) Using a pressure cuff, locate a suitable vein in the arm.

(ii) Cleanse thoroughly the skin over the vein using tincture of iodine followed by ethanol/ether.

(iii) Remove the protective cap from the top of the culture bottle and cleanse the top of each bottle using an ethanol/ether swab.

(iv) Using a sterile syringe and size 2l/suitable gauge needle withdraw 10 ml of blood.

(v) With care, remove the needle from the syringe and dispense the blood into culture bottle in the vicinity of a flame.

(vi) Using an ethanol/ether swab, wipe the top of culture bottle and replace the cap. Gently mix the blood with the broth (Blood/broth volume should be atleast 1:10).

N.B.: The blood must not be allowed to clot in the culture media because any bacteria will become trapped in the clot.

(vii) Label the bottle with name, registration number of patient, ward, unit, date and time of collection.

(viii) As early as possible, incubate the inoculated media at 35 ° — 37° C.

 

INCUBATION

Incubate at 35°C — 37°C for upto 7 days with regular examining and subculturing as per guidelines.

 

SIGNS OF BACTERIAL GROWTH

Examine daily for upto 7 days.

(i) Turbidity above the red cell layer.

(ii) Haemolysis of the red blood cells.

(iii) Gas bubbles.

(iv)Appearance of small colonies in the broth on the surface of sedimented red cell layer or along the wall of bottle.

A sterile culture usually remains clear. A slight turbidity may develop after several days of incubation. 

lf there are signs of bacterial growth, subculture the broth and examine a Gram stained smear for bacteria and then do standard follow up for identification of organism.

 

BACTEC

• The BACTEC blood culture system is a fully automated microbiology growth and detection system designed to detect microbial growth from blood specimens.

• The BACTEC™ FX and the BACTEC™ 9000 family of continuous monitoring blood culturing instruments offering performance, safety, reliability, ease of use, media quality and service.

• The BACTEC 9000 series of blood culture instruments are designed for the rapid detection of microorganisms in clinical specimens. The sample to be tested is inoculated into the vial which is entered into the BACTEC instrument for incubation and periodic reading.

• Each vial contains a sensor which responds to the concentration of CO2 produced by the metabolism of microorganisms or the consumption of oxygen needed for the growth of microorganisms. 

• The sensor is monitored by the instrument every ten minutes for an increase in its fluorescence, which is proportional to the increasing amount of CO2 or the decreasing amount of O2 present in the vial. 

• A positive reading indicates the presumptive presence of viable microorganisms in the vial.

 

COLLECTION AND TRANSPORT OF VARIOUS BODY FLUIDS

 

COLLECTION AND TRANSPORT OF EFFUSIONS

• Synovial, Pleural, Pericardial, Ascitic and Hydrocele fluids
• An effusion is fluid which collects in a body cavity. Fluids which collected due to an inflammatory process is referred as an exudate and that which forms due to a noninflammatory process is referred as a transudate.

• Collection and transport of effusions:
• After aspiration, aseptically dispense the fluid as follows:

• 2 — 3 ml into dry, sterile, screw — cap tube or bottle.

• 9 ml into a screw — cap tube or bottle which contains l ml of sodium citrate solution. Mix the fluid with the anti - coagulant.

• An anticoagulant is required to prevent clotting, especially exudates. The citrated samples can be used for the cell count, protein estimation, microscopy and culture. A sample without citrate is useful to see whether clotting occurs.

• Label it properly and deliver the specimen with a request form to the laboratory as early as possible.

• If there is delay in transport of specimen, then dispense 5 ml of fluid into a bottle of sterile thioglycollate broth and mix.

 

COLLECTION AND TRANSPORT OF CSF

• Cerebrospinal fluid must be collected by an experienced Medical Officer or health worker. It must be collected aseptically to prevent organisms being introduced into the central nervous system. The fluid is usually collected from the arachnoid space.

• A sterile wide bore needle is inserted between the fourth and fifth lumbar vertebrae and the CSF is allowed to drip into a dry sterile container. A ventricular puncture is sometimes performed to collect CSF from infants.

• CSF should be examined as early as possible. A delay in examining CSF reduces the chances of isolating a pathogen. It will also result in a lower cell count due to WBCs being lysed, and to a falsely low glucose value due to glycolysis.

• When trypanosomes are present, they will be difficult to find because they are rapidly lyzed once the CSF has been withdrawn.

 

COLLECTION OF CSF

1.    Take two sterile, dry, screw-capped containers and label one No.1 (first sample for culture purpose) and No.2 (second sample for other investigations).

2.    Collect about 1 ml of CSF in container No.1 and about 2-3 ml in container No.2.

3.    Immediately deliver the samples with a request form to the laboratory.

COMMON PATHOGENS IN CSF

BACTERIA	GRAM POSITIVE: Streptococcus pneumoniae, Streptococcus agalactiae, Listeria monocytogenes.
	GRAM NEGATIVE: Neisseria meningitides, Haemophilus influenza type b, Escherichia coli, Pseudomonas aeruginosa, Proteus species, Salmonella species,Flavobacterium meningosepticum
OTHER BACTERIA	Mycobacterium tuberculosis, Treponema pallidum
FUNGI	Cryptococcus neoformans (mainly in AIDS patients) and less commonly Aspergillus species
VIRUSES	Coxsackieviruses, echovirus, and arboviruses, herpes simplex 2 virus, varicella zoster virus and Lymphocytic choreomeningitis virus (LCM). Rarely polio viruses.
PARASITES	Trypanosoma species and Naegleria fowleri. Also Toxoplasma gondii (in AIDS patients)