This hospital uses the Biomerieux BacT/ALERT 3D colorimetric detection system for processing blood cultures. The carbon dioxide (CO2) produced by bacterial growth changes the colour of the colorimetric sensor at the base of each bottle and alerts the biomedical scientist to the presence of potential bacterial growth.
The system’s efficiency depends on the quality of the sample. Here are some tips to ensure a good quality test result.
1. Equipment preparation
2. Patient preparation
3. Sample collection
These bottles contain a larger volume of 30ml of clear liquid media which contain absorbent polymeric beads. This formulation is designed to enhance the performance of the culture only. Storage of pre-inoculated bottles remain the same: store at room temperature away from direct sunlight. Inoculation procedures also remain the same: inoculate with 4ml of patient blood and transport to the microbiology without delay.
The detection of Chlamydia Trachomatis (CT) and/or Neisseria gonorrhoea (NG) DNA in specimens is carried out using the Cobas 4800 CT/NG Test. This is a type of PCR system.
Note: Do not discard the media inside the tube.
Please ensure swabs are broken at the score line, incorrectly processed samples may cause technical problems.
If the urine is not filled to between the two lines we can not test the sample and a repeat sample will be asked for
Any samples received with 2 or swabs in each container or no swab at all will not be processed.
Please make the request form clear as to whether Chlamydia (CT) or Gonorrhoea (GC) testing is required. Gonorrhoea testing is currently only available through GUM.
The laboratory will only ever test what is requested on the form.
Rectal, throat, urethral and Eye swabs are not validated for testing. These can be tested but will have the comment “Specimen not validated for testing by Roche Cobas”
Clinical details required:
Clinical details required:
Pernasal swabs are used for the detection of Bordetella species (whooping cough). These are special wire swabs designed to reach the pernasal area, they can be obtained from email@example.com. We do not process charcoal/aimes swabs for whooping cough
If an outbreak is suspected please notify the local health protection unit prior to sending swabs.
Generally throat swabs are used to isolate ß-haemolytic Streptococci, specially A, C and G. If appropriate clinical details are given then a wider range of investigations can be initiated. Organisms such as Fusobacterium necrophorum, Arcanobacterium haemolyticum and Corynebacterium ulcerans are more unusual causes of sore throats so these are not routinely screened for unless significant clinical details are provided.
Clinical details required:
If a sexually transmitted infection (Gonorrhoea) is suspected please indicate on the request form
Clinical details required:
All eyes swabs are cultured using the same method. Please include past and current antimicrobial therapy, history of previous infections and any information about specific clinical details which may help our investigations.
All neonatal eyes swabs will be screened for Gonorrhoea. If gonorrhoea is suspected in any other patient, please indicate on the form
Examples of clinical details required:
Please note that faeces culture is a highly specialised series of tests that involves screening a sample for a number of bacterial pathogens causing gastroenteritis. Therefore our laboratory requires clinical information to select the appropriate tests. Please include as much information as possible regarding:
The department requires a minimum of 5ml of faeces collected into a 30ml sterile universal or blue top with 'spoon' for processing. Please note also that samples should be fresh (ideally sent up to the laboratory within 2-3 hours of being passed).
If faeces contaminated with urine then M, C & S, parasitology and clostridium difficile investigations can be tested but this may affect the result.
Culture for enteric pathogens includes: campylobacter species, salmonella species and E. coli O:157 in all cases of non-hospital acquired diarrhoea. The department also screens for shigella species in all patients with a history of foreign travel. We also screen for vibrio cholerae in patients with a history of travel to cholera endemic areas AND if the patient has a liquid stool. We screen for Vibrio parahaemolyticus in patients with a history of shellfish consumption or participation in water related activities. Lastly, the department also cultures for Yersinia enterocolitica in patients with symptoms suggestive of yersiniosis (especially young or elderly patients with mesenteric lymphadenitis, pseudoappendicitis, terminal Ileitis).
Samples will not be cultured unless appropriate clinical information is provided. The microbiology department deals with a large volume of samples; therefore it is vital that sufficient clinical details are provided to establish the cause of diarrhoea as being unrelated to C. difficile.
All GP samples and the following wards/ departments will receive faeces culture (If requested) regardless of clinical Information.
Emergency department, MAU, ICU, SAU, CDU, all paediatric wards, all outpatient wards/ clinics wards 305 and 304. The microbiology department wil also check samples from other inpatient wards where culture has been requested, for admission dates. Samples from patients who have been in hospital for less than 3 days will also be cultured.
What clinical details should I provide?
Travel to foreign countries may result in infectious diarrhoea being attributed to more unusual pathogens not seen (or seen rarely) in the UK. Faeces foreign travel investigation includes additional culture methods and additional microscopyfor faecal parasites. It is vital that if a patient has been abroad to provide as much information as possible such as:
Where has the patient been?
How long ago?
How long have they had their symptoms (this is especially important for determining what tests are performed, as protracted diarrhoea is a common feature of infection with intestinal parasites).
Is the patient immunocompromised?
If a High Risk pathogen is suspected, please indicate so we can process the sample using the correct safety procedures.
Which parasites will the department screen for?
In the laboratory, provided the relevant clinical details are given, we will look for Helminths (worms) e.g. Ascaris, hookworm, tapeworm, liver flukes and other Helminths. We will also look for Protozoan parasites e.g. Giardia, Cryptosporidia, Entamoeba and others.
What happens if I don’t provide suitable clinical information?
Please note that samples will not be examined for ova, cyst’s and parasites unless appropriate clinical information is provided. The microbiology department deals with a large volume of samples; therefore it is vital that in these cases, sufficient clinical details are provided to establish the cause of diarrhoea as being unrelated to C. difficile.
When will the sample be tested?
The microbiology department currently processes faeces samples for C. difficile investigations on a 7 day basis.
Monday - Friday: 2 runs are performed in the day (am and pm). Samples must reach pathology reception by 9am at the latest for the morning run and 12.30pm for the afternoon run.
Saturday - Sunday: 1 run is performed each day. Samples must reach pathology reception by 9am at the latest.
C. difficiie is an opportunistic anaerobic spore forming bacteria present in the intestines. It can produce toxins which results in infection. Some strains do not produce toxins but the bacteria being present can mean that the patient has the potential to excrete it and have an increased risk of developing the infection and diarrhoea.
This method will differentiate between patients who have active C. difficile infection and those that could be at risk of becoming infected (excretors).
This new method aims to provide more effective and consistent diagnosis across the NHS. The 2 stages include:
ICM users will now see the test as:
Faeces (C. difficile screen only)
The is no change for other electronic users. IF an electronic ordering system is not available please request 'C. diff screen'.
A minimum of ¼ of a pot of faeces of Bristol stool type 6 or 7 must be submitted for testing.
Samples must be sent immediately (within 2 hours) to the lab (a delay in transport can result in a significant loss of any toxin that may be present in the sample). Samples that cannot be processed within two hours must be refrigerated (up to two days).
Please consult Infection Control for guidance as to when to submit samples per episode of diarrhoea.
Please be aware of our criteria for not testing of stool samples for C difficile toxin.
* Samples may be processed but only after clearance from a consultant microbiologist or the Infection Control Team.
What are the indications for testing a sample for enteric viruses?
Most people with viral gastroenteritis have only mild symptoms, and the condition improves within a few days without the need for treatment. However in certain cases, for example; if the symptoms are severe, persistent or recurrent and if the patient is especially vulnerable due to age or immune status, or in outbreaks of diarrhoea it may be clinically useful to isolate the causative agent
What type of viral investigations are carried out routinely?
The microbiology department only carries out in-house testing for rotavirus in paediatric patients (<5 years). Norovirus testing is also carried out in outbreaks of diarrhoea and vomiting after notification by the Infection control team only.
How do I go about requesting virology investigations on individual patients that are not under 5 or part of an outbreak of diarrhoea and vomiting?
Any requests for testing of patient samples, which do not meet the above criteria, must be approved by a consultant microbiologist. This can be done by contacting the on-call Consultant microbiologist through switchboard 01332 340171. Please have the patient’s details ready. Any samples submitted for virology thereafter must state on the request form that testing has been approved by a consultant microbiologist.
What happens if I don’t get approval from a consultant microbiologist?
Please note that samples will not be examined for enteric viruses unless approved by a consultant microbiologist. The microbiology department deals with a large volume of samples and aside from Rotavirus, samples have to be referred for viral investigation.
Skin and nail samples should be collected into a Dermapak. If these are not available then a sterile universal may be used. Please do not use non sterile packets or sellotape as the culture may fail due to contamination with environmental fungi.
Skin swabs will NOT be processed for full fungal investigations.
Please provide one request form per sample. A Dermapak is not a suitable request form.
Please include the following information if relevant:
If Pityriasis versicolor is suspected please note clearly on the request form as this will significantly affect the report. This organism cannot be grown using conventional culture techniques and so the microscopy is heavily relied upon.
Storage of specimens
Skin and nail specimens should be stored at room temperature, as dermatophyte growth may be compromised between 4 - 6 oC.
Only skin, hair and nail samples will be investigated for fungi (dermatophytes and saprophytes) this can take up to 3 weeks. If Candida is suspected ensure this is written in the clinical details. The following will always be investigated for Candida:
A microscopy result will be issued within 24hrs of receipt of sample (Mon-Fri). The culture can take up to 3 weeks to grow. Calcofluor white is used for the microscopy, it is a fluorescent stain that highlights any fungal elements present in the sample.
The microscopy result will identify presence of fungal elements whether viable or not. The culture result being negative even with a positive microscopy as different pieces of skin and nail are used for each test.
Potentially significant saprophytic moulds will be reported with an explanation as not they will not always be the cause of the infection, this will be dependent on the clinical history and presentation of the patient.
In patients with suspected tinea or ringworm the area should be cleaned with an alcohol wipe (70% alcohol) before taking the sample. This will remove any potential contamination and ointments.
Using a sterile blunt implement, scrape the lesion, particularly at the advancing edge. If there are multiple lesions choose the most recent for testing as the older the lesion the less likely that viable organism will be present. Please send plenty of scrapings as at least 9 pieces are required for a full microscopy and culture.
It is vital that good quality samples are sent to the laboratory for testing as the results could be misleading and result in a false negative report.
If possible, collect the sub-ungual debris in addition to nail clippings. Take pieces of the discoloured, dystrophic or brittle parts of the nail only, sampling as far back as possible from the distal part of the nail. At least 9 pieces are required for a full test. If there is insufficient then only a microscopy will be carried out.
Only send plucked hairs from the affected area along with some scalp scrapings. Cut hair is not suitable as the infection tends to affect just near or below the surface of the scalp. Please send at least 8 pulled hairs for testing.
All vaginal swabs will receive a microscopy to identify yeast cells, bacterial vaginosis (BV) and Trichomonas vaginalis (TV). Culture for Candida is also carried out on all swabs in this group.
Please note that vaginal and vulval swabs are not appropriate specimen types for Gonorrhoea investigations.
The only exception to this are paediatric patients where vaginal swabs will be investigated for Gonorrhoea if the clinical details are suggestive of sexual assault.
Please provide the below clinical details so that further bacterial culture can be carried out:
How to collect a vaginal sample/ patient preperation
Cervical and urethral swabs will be investigated for Neisseria gonorrhoea ONLY. So, please ensure an HVS is also taken to ensure all possible causes of infection are tested for.
How to collect a cervical sample/ patient preperation
For urethral swabs, Candida investigations on catheterized & elderly patients can be performed, but only if requested.
Routine culture will be performed on all penile swabs so please request M, C & S superficial swab. DO NOT request genital screen.
Penile swabs are routinely cultured for causes of general skin and soft tissue infections e.g. Staphylococcus aureus, Beta haemolytic Streptococci, Candida etc.
Neisseria Gonorrhoea will ONLY be screened for if the clinical details suggest this as a cause of infection.
Please see separately.
MRSA screening swabs are taken to ascertain whether the patient carries methicillin resistant Staphylococcus aureus (MRSA).The hospital carries out screens on elective, high risk, emergency patients and routine MRSA screens.
The screening sites for routine screens are the same as those for emergency admission screens. In addition, a line tip must be sent to microbiology, in addition to invasive device site swab only if infection is suspected via an invasive device.
Neonates from other hospitals:
Ensure that the swab/s being used are in there use by date.
When taking nasal swabs, swab both nostrils with the same swab.
It is recommended to moisten the swab with sterile water or sterile saline prior to swabbing (not essential)
The optimal time for collection is before MRSA treatment has commenced.
MRSA screening swabs are cultured 7 days a week. If there is a delay in sending swabs to Pathology, refrigeration is preferable to storage at ambient temperature. Delays of over 48 hours are undesirable.
NB. Samples received without the site or investigation stated, will not be tested
The following labels (attached to request forms) denote the type of patient.
Blue Label: Admitted as an Emergency.
Yellow Label: Elective Admission screen.
No label: Routine screen.
Please ensure that the correct stickers are firmly affixed to the patients request form. This system allows for coding of patients to ensure ward compliance with the hospital screening policy.
Ensure that the stickers are on each form if sending more than 1 sample.
For infection control purposes it is vital that the exact location of the patient is specified.
When ordering MRSA screening on ICM, ensure one form is ordered for every sample, specifying each sample site separately (if more than one sample taken from a patient).
If using a hand written form ensure all details are filled in correctly e.g. location, sample site etc.., taking special care to tick the MRSA screen box (if pre-op) or writing MRSA in other.
It is possible to order to add MRSA to another investigation. Select the culture required and add MRSA as an additional test
Please include any details that show why Mycobacteria spp is suspected especially ‘atypicals’ as these samples may need to be handled differently especially with skin related infections.
Tuberculosis is caused by Mycobacterium tuberculosis complex; a group of acid fast bacilli (AFB). These bacteria are very slow growing and can take up to 8 weeks for the laboratory to isolate them. Once isolated, they are sent to the reference laboratory for full identification and antibiotic susceptibility testing.
This laboratory uses the BacT/Alert 3D Mycobacterial liquid culture system to isolate Mycobacteria spp. It uses a colorimetric sensor and reflected light to monitor the presence and production of Carbon dioxide (CO2). If micro-organisms are present in the test sample CO2 is produced which affects the gas-permeable sensor at the bottom of each bottle. The change in colour results in an increase in reflectance units which causes the system to alert us to a positive bottle.
All samples are screened for the presence of AFB's using a fluorescence stain (auramine). These are reported promptly by the consultant microbiologists.
This method is suitable to enable growth of most types of atypical Mycobacteria but some have slightly different growth requirements; because of this it is vital that the laboratory is informed of any clinical details which may lead us to investigate further.
Ensure that at least 2 ml of sample is sent to us in a sterile universal. If additional tests are required such as routine culture, please send a separate sample where possible.
It is important to send 3 sputum samples on consecutive days; this will increase the chances of isolating any Mycobacteria spp that may be present.
For more information regarding alternative testing methods, treatment or epidemiology please the TB nurses in the Royal Derby hospital or the on-call consultant microbiologist.
When the cough is dry, physiotherapy, postural drainage or inhalation of nebulised saline before expectoration may be helpful.
Broncho-alveolar washing/bronchial washings
Minimum sample size is preferably 5 ml.
Gastric washings (for children)
Collect samples early in the morning (before breakfast) on 3 consecutive days.
A minimum volume of 5 ml should be provided.
Aspirates should be promptly delivered to avoid acidic deterioration of organisms.
Results of direct microscopy on gastric washing can be misleading as acid-fast bacilli are normally present in the stomach.
Sterile Body Fluids (CSF, Pleural Fluids)
CSF- Collect aseptically as much CSF sample as possible into a sterile container. If a small volume is available after initial lumbar puncture and the findings of cell counts and protein suggest TB meningitis, a second procedure should be considered to obtain a larger volume to improve chances of achieving positive cultures.
Pleural Fluids - Ensure that at least 2 ml of sample is sent to us in a sterile universal. If additional tests are required such as routine culture, please send a separate sample where possible.
Tissue and Pus
Tissue - Send 1-4 small tissue pieces ideally 1cm3 in size.
Pus - A minimum volume of 2 ml should be provided.
Collect in the entire 1st early morning produced on three consecutive days in a large early morning urine container.
Sample should be added directly to a sodium citrate bottle if liquid or a sterile universal if solid.
Ensure the wound is cleared of debris and cleaned as appropriate before taking the sample. If necrotic/ overtly pussy areas are swabbed the results can be of little diagnostic value – ‘mixed colonising bacteria present’
If insufficient clinical details are given, the laboratory cannot always investigate the sample fully to ensure that all potential pathogens are looked for. Please include any relevant information such as:
Clinical details required:
If the sample is from an intensive care setting than we will automatically look for MRSA, Coliforms and Candida. If these are ever suspected please request them specifically giving appropriate reasons.
It is important to inform the laboratory of clinical information such as:
These details mean that the sample may be treated slightly differently to the routine ones or they could be cultured to ensure a wider range of organisms are looked for that are specific to these types of patients
If no clinical details are given then a basic culture is performed, this is usually sufficient for most routine patients but may be detrimental particularly to those with chronic/ systemic infections.
Cystic fibrosis patients are at a higher risk developing respiratory tract infections. This group of patients are more susceptible to infections caused by Pseudomonas spp, Staphylococcus aureus, Candida spp, fungi and in severe cases Burkholderia cenocepacia complex. These organisms are targeted in our culture methods so long as we are informed on the request form the patient has cystic fibrosis.
Salivary samples are not of suitable quality for culture as the oral normal flora can overgrow the pathogens therefore resulting in a ‘negative’ culture.
We need at least 2ml of sample in a sterile universal. If other tests are required such as virology/ PCP, please send a separate sample where possible and include all significant clinical details on the request form.
Cough swabs are not suitable samples but can be used only in children with cystic fibrosis.
A ‘mid-stream’ urine (MSU) sample is necessary for aculture so it is important to ensure that the sample is collected aseptically. The procedure for collecting a clean ‘mid-stream’ includes the following steps:
Uncontaminated specimens can also be obtained from catheterised patients following the same hygienic procedures for the end of the catheter.
Bag urines are only excepted on Neonates, please do not collect bag urines on adult patients.
Suprapubic Aspirates and Nephrostomy Urines must be labelled as the relevant sample type. Collection of these samples types must be by trained staff only.
Each urine sample is processed microscopically using an automated machine. Depending on the clinical details and microscopic results the urine may then be cultured onto Chromogenic agar and incubated overnight.
All urines that are considered negative by microscopy will not receive a culture and the result will be authorised immediately.
The table below contains the clinical decision values behind what urines will be cultured. The top 4 highlighted sections will all receive a culture regardless of the microscopy result. It is therefore paramount that this type of information is provided on the request form.
|Clinical details||AN or Renal Transplant|
|Location||Ward 302 ,303 and 314|
|Specimen type||UN (Nephrostomy urine) or USP (Supra pubic aspirate urine)|
|Seen||If Yeast or Trichomonas|
|Polymorph count||PC >7000|
All microscopy positive urines will be cultured over night.
Bordetella pertussis is the organism that causes Whooping cough. It is a fastidious organism that can take up to 7 days to grow. These organisms are then confirmed by reference laboratories.
Per nasal swabs are used for the detection of Bordetella species (whooping cough). These are special wire swabs designed to reach the per nasal area, they can be obtained from firstname.lastname@example.org. We do not process charcoal/aimes swabs for whooping cough
If an outbreak is suspected please notify the local health protection unit prior to sending swabs.
Insert the swab into the nostril and guide it gently and horizontally to the back of the nose. If an obstruction is encountered, withdraw the swab and reinsert through the other nostril.
As soon as the resistance to the posterior pharyngeal wall is felt, withdraw the swab and insert into transport medium.
Send the swab in transport media directly to the laboratory.
Notifying Public Health England is the responsibility of the requesting clinician.