Isolating and identifying bacteria (human health)

Introduction

In this course you will learn about microbiology techniques for identifying bacteria, focusing on WHO priority pathogens which are the focus of the Global Antimicrobial Resistance Surveillance System (GLASS). You will start by looking at the diverse types of clinical samples sent to microbiology laboratories and factors that can impact the testing of samples and reporting of test results. Basic tests used routinely in laboratories to isolate and identify bacteria are discussed next, followed by newer and/or more advanced methods usually performed by reference laboratories. Finally, you will be introduced to Quality Control (QC) measures that should be implemented in the clinical microbiology laboratory to ensure that test results are reliable and accurate. You will learn more about quality measures in the Quality assurance and AMR surveillance course.

Basic knowledge of concepts such as bacterial growth and bacterial structure is assumed. If you are unfamiliar with these concepts or would like a refresher course, you might want to look back at the Introducing antimicrobial resistance course or this Microbiology textbook (OpenStax, 2021). Information about the GLASS programme can be found in the Introducing AMR surveillance systems course.

After completing this course, you will be able to:

• know where samples are obtained from and reflect on the process by which bacterial samples are processed in your workplace
• describe the principles of laboratory tests used to isolate and identify key bacterial pathogens in human health, which are the focus of the GLASS programme
• know when and why advanced testing such as mass spectrometry and automated systems are used
• know the importance of procedures designed to ensure the quality of laboratory work relating to isolating and identifying bacteria in your workplace.

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1 Principles of sampling and specimen collection

In this section we introduce the basics of sampling and how to collect and process clinical specimens.

1.1 Origins of samples

Clinical samples come from a range of different settings, such as:

• secondary and tertiary care patients in hospital
• community patients seen in clinics
• specialist community clinics and outpatient departments, for example tuberculosis (TB) or sexual health services.

Figure 1 Clinical swabs.

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Samples also showing packaging and request forms: blood cultures, urine, pus and swabs.

Figure 1 Clinical swabs.

Clinical samples are generally sent to microbiology laboratories to guide antimicrobial therapy (Figure 1). This is done by identifying which pathogens are present in the sample by culturing them. These pathogens can then additionally be tested for antimicrobial resistance (AMR).

Clinical specimens are also routinely used as a source of data for AMR surveillance. However, a significant proportion of patients who could have a microbiology test performed never get sampled and so no results are available for them. This introduces an element of bias into the data. As a consequence, more severe cases and treatment failures could be over-represented. Tertiary care cases could also have more samples sent than those in more remote settings which could affect the overall results if resistance patterns differ between the settings. For long term, sustainable AMR surveillance, however, it is much cheaper and simpler to use clinical samples and the associated data than establishing a study to sample more randomly. The data are also always available whereas a surveillance study might be a one-off event.

Whilst it is not possible to avoid all potential bias when using clinical samples for surveillance purposes, it helps to be aware that reported rates of AMR may not be representative of the country or region as a whole.

1.2 Impact of sample quality on testing and reporting of results

Not all samples received by a laboratory will be of high quality, that is, contaminant free and in sufficient quantity. Prior to starting the testing process, laboratory staff should be aware of potential quality issues relating to specific sample types that could lead to false results.

Reducing contamination

Clinical staff can take several measures to improve the quality of the specimens they send to laboratories, either by reducing contamination through aseptic techniques and/or increasing the yield of true pathogens. For example, sterile containers and equipment should be used for sample collection, to avoid the introduction of contaminating organisms. Specimens need to be large enough to handle effectively and to minimise changes such as pH, drying out, etc., that may affect the viability of the organism concerned.

Some types of sample may require special treatment or have other considerations.

• Sputum samples, for instance are very useful for diagnosing TB and are frequently sent to diagnose other respiratory tract infections. However, the results have to be considered carefully as the sample is likely to be contaminated with the commensal flora in the upper respiratory tract. Identifying all the organisms in a sputum sample will not help the diagnosis and takes up a lot of laboratory time and resources.
• Blood cultures, which are important in diagnosing severe and/or bloodstream infections, are another sample type that is easily contaminated by the commensal flora of the skin. Even though the blood is taken carefully to minimise contamination and inoculated directly into blood culture media by the person taking the samples, contaminants will still sometimes be found. A video showing safe and accurate blood culture collection using ANTT (The Hillingdon Hospitals NHS Foundation Trust, 2019), shows how careful decontamination of the skin and bottles, plus aseptic technique can minimise sample contamination. Video 1 illustrates some of the key features of aseptic technique.
• Urine samples need to be collected carefully in midstream, to help reduce contamination with the commensal flora in the genital region. The sample should be refrigerated if it cannot be processed immediately. Boric acid can be used as a preservative if this is not possible. Microscopy helps determine whether a urinary tract infection (UTI) is present before culturing by looking for pus cells which indicate inflammation/infection.

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Transcript: Video 1 Aseptic technique

Microbes are grown in the laboratory as cultures. Cultures can be grown from samples from a variety of sources including clinical specimens. In their natural environments, many species of bacteria grow together, but if we want to study a particular species, we need to grow it on its own in a pure culture. In a pure culture, all the cells present are of the same type. Pure cultures are maintained by sterilising media before use and by a practice called ‘aseptic technique’.

Aseptic technique prevents other microbes contaminating our culture and also prevents cultured microbes from escaping into the environment, which is very important when we’re dealing with pathogens.

To grow microbes, we must supply all their nutritional requirements by growing them in culture media. Media can be liquid, when they are called broths, or they can be solidified with a jelly-like substance derived from seaweed, called agar.

A wide range of different media are available as different bacteria have different nutritional requirements. In fact microbes can sometimes be identified by the media they can or cannot grow on.

Media must be sterilised before use and this is normally done in an autoclave, a type of industrial pressure cooker. Materials are sterilised by exposure to high pressure steam at a temperature of over 121 °C for 15 minutes. Sterilising media before use means that the only cultures that grow are the ones we put there.

To check whether sterility (the complete absence of living organisms) has been achieved, the materials are marked with special tape that changes colour under these conditions.

To prevent the spread of any pathogens into the environment, bacterial cultures that are not required any more are sterilised by autoclaving.

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Video 1 Aseptic technique

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Laboratory staff also need to take care not to introduce contaminants into cultures. Good laboratory practice should be followed at all times.

1.3 Transport, labelling and processing of samples

Specimens should be sent to the laboratory as quickly as possible so that the causative agent is still viable (able to replicate and divide) when it gets there, and is not overwhelmed by the growth of commensal organisms or contaminants. A variety of techniques are used to achieve this aim and to minimise the effects of any delay. Refrigeration and carefully designed transport media can keep the organisms viable for longer but add to costs.

Each sample should be uniquely labelled and registered in a database so that it can be attributed to a specific patient, and its analysis prioritised according to the urgency with which the result is required. It is important to make sure results are stored securely to protect the patient’s confidentiality.

Not all testing of isolates can be carried out in a hospital laboratory and specimens/cultures may need to be sent to a reference laboratory for further processing. For example, some organisms may be difficult to culture or may need more advanced techniques to identify to species level (see Section 3.3).

2 Basic tests used to isolate and identify bacterial pathogens

We turn now to a description of the laboratory tests used to identify pathogens.

2.1 Culture media

Growing isolates from clinical samples in the laboratory is a useful part of diagnosing the cause of an infectious disease and is an essential step for determining the antibiotic sensitivities of bacteria.

All artificial media comprise a mineral base, a nitrogen source, a carbon source and an energy source. Supplements of organic compounds may be added to encourage or suppress the growth requirements of particular microorganisms. Media may be in liquid (broth) or solid form.

Most clinical samples are inoculated directly onto solid, agar-based culture media on a plate. Videos 2 and 3 demonstrate two common methods for preparing plates, streak plating and spread plating.

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Transcript: Video 2 Streak plating

All bacteria in a single colony are usually identical. In a pure culture all the colonies will be alike in shape, size, colour, texture and smell. But in a mixed culture, like a clinical specimen, several different types of colony may be seen.

To check whether a culture is pure we use a technique called streak plating.

The first step is to label the plate with the date and the identity of the sample.

Labels are written on the part of the plate containing the agar so that if the lid is lost the culture can still be identified.

The Petri dish is kept upside down to minimise the chances of a contaminant falling onto the agar.

To make a streak plate we use aseptic technique and a bacteriologist’s loop.

A bottle containing the specimen is taken, flamed to remove any contaminating organisms, and the loop used to sample the microbes within. The loop full of microbes is touched to the surface of the agar and is spread across the plate in a series of streaks that act to successively dilute out the sample.

The plate is turned between each set of streaks. The final streak, a wiggly line, should allow individual microbes to be separated.

In order to more clearly see the streaking technique, we’ve drawn the pattern of streaks on the bottom of a Petri dish, so you can see the order in which they are made.

Bacteria that can grow in the human body are incubated at 37 °C.

After overnight incubation, at the more dilute end of the streak series, separate colonies, each resulting from the growth of a single microbe can be seen.

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Video 2 Streak plating

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Transcript: Video 3 Spread plating

Using aseptic technique, we take out 0.1 ml of a particular dilution from its tube and place it onto the middle of the agar surface.

The inoculum is spread over the plate using a spreader. This one is pre-packed and sterilised. The spreader is moved over the surface of the agar, while the plate itself is rotated with the other hand to give an even spread of bacteria across the surface.

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Video 3 Spread plating

When grown at optimal temperatures in suitable conditions, this process enables the laboratory technologist to view individual colonies and select them for additional work according to their appearance (colonial morphology) and relative abundance. Table 1 provides details of the main media types used routinely in microbiology laboratories. Note that media may fall into more than one of the categories listed, for example it may be both selective and indicator.

2.2 Processing blood cultures

Blood cultures are inoculated directly from the patient into sterile liquid media. These media may be locally made, or commercial culture bottles can be purchased. The bottles are incubated in the laboratory, observed and sub-cultured. They are plated out onto agar plates for further work to determine if the blood culture is positive. Sub-culturing on a mix of media types is necessary to isolate most of the organisms likely to cause bloodstream infections.

There are also a number of automated systems in common use, such as BD BACTEC^TM, or BacT/ALERT® (Biomerieux) which use pre-prepared bottles (Figure 2). These can monitor the samples for growth and indicate when it is time to do the sub-culture. Although the equipment is expensive, the risk of contaminants being introduced is reduced and turnaround times are usually faster too, as the subculture can be set up as soon as the equipment indicates positive growth, instead of once daily as is usual for manual blood cultures.

Figure 2 Removing a positive bottle from an automated blood culture machine

2.3 Gram stain

The Gram stain test allows you to see basic bacterial morphology and group bacteria into two main types; Gram-positive and Gram-negative.

Watch Video 4 to see how the test is performed. If you are unable to watch this video, you can view a transcript of the content by clicking on ‘Show transcript’ below.

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Transcript: Video 4 How to perform a Gram stain

The Gram stain is the most important stain in microbiology. The first step is to heat-fix the bacteria to a microscope slide. Using a sterile loop, a few drops of water are transferred to the slide. The culture is then mixed with the water to give an emulsion and this is spread onto the slide. The secret of a good Gram stain is not to make the emulsion too thick. The emulsion is left to dry at room temperature.

Then the slide is passed through the Bunsen flame to fix the bacteria to the glass, so that they won’t be washed off by the staining. It’s important not to let the slide get too hot or the bacteria will burst. If it’s too hot for you, it will be too hot for the bacteria.

In Gram’s staining, the slide is flooded with crystal violet stain and left to stand for one minute. The crystal violet is washed off with slow-running tap water. The next step is to flood the slide with Gram’s iodine, which is also left for a minute. The iodine forms a complex with the crystal violet, giving a dark purple colour. The slide is rinsed again before the next step.

Next, 95% alcohol is used to wash the purple dye away. Some bacteria retain the dye; others don’t. After a rinse in water, the final step is to add a red dye, Safranin, which is left on for just 30 seconds.

After a final wash, the slide is blotted, dried, and then examined under the microscope. Some bacteria appear pink. Since they lost the original purple colour, they are called Gram negative. Other bacteria retain the purple colour, which masks the red dye, and these ones are called Gram positive. Round bacteria are called cocci. Rod-shaped bacteria are often known as bacilli.

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Video 4 How to perform a Gram stain

Note that laboratories without a gas supply can still perform Gram stain tests by using single use sterile plastic loops which are disposed of into disinfectant and the slide may be fixed by flooding with methanol (a technique that is preferred in some cases anyway).

3 Commercially available and more advanced tests

Once the specimens have been cultured and single colonies are available growing on a plate, additional testing is done to determine the bacterial species. Many laboratories rely on standard biochemical testing. Biochemical test strips are available which enable multiple tests to be performed at the same time.

3.1 Biochemical test strips

Multiple biochemical tests in a single kit form, come as a strip with micro wells and reagent. The testing process is straightforward; inoculate, incubate, add some more reagents then read and compare the result to a standard. Examples of testing kits include Analytical Profile Index (API, Biomerieux) and Microbact (Thermofisher).

Activity 7: API identification of Gram-negative pathogens

Timing: Allow 15 minutes

In this activity you will first watch a video showing how to set up an API test for the identification of Gram-negative pathogens, and then answer some related questions.

First, watch the video ‘Performing an API20E strip test’ (2016).

Note that in the video Enterbacterales are referred to by the old term ‘Enterobacteriaceae’.

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Transcript

Add water to 3/4 fill all of the honeycomb wells of the tray

Flame a loop until it is red hot then allow it to cool for 20 seconds

Select a single isolated colony and emulsify in 10 mils of sterile distilled water

Use a new sterile pipette

For CIT, VP and gel tests fill both the tube and cupule

Fill only the tubes for the remaining tests

Add one or two drops of paraffin oil two tests: ADH, LDC, ODC, H2S and URE

Incubate the strip at 37°C overnight

After incubation add biochemical reagents to the required tests

One drop each of VP1 and VP2 to the VP test

One drop of Kovacs reagent to the IND test and

One drop of the TDA reagent to the TDA test

After 10 minutes compare the colours against the chart and record on an API record sheet

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Now, answer the following questions:

1. What temperature is the test kit incubated at overnight?
2. Would the same API kit be suitable for all of the following: E. coli, Shigella, Salmonella, Klebsiella, Haemophilus influenzae?

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Discussion

1. The test kit should be incubated at 37°C overnight.
2. No. ‘E’ in API20E stands for Enterobacterales (formerly known as Enterobacteriaceae), so all of these organisms are identified by API 20E except Haemophilus influenzae. Haemophilus influenzae, as a non-glucose fermenter, is not classified as Enterobacterales. A different kit (API 20NE – ‘Non-Enterobacterales’) would be needed to identify Haemophilus influenzae.

3.2 Chromogenic media

Chromogenic media, first mentioned in Section 2.1, are useful in certain situations, for example to identify:

• organisms in potentially mixed samples – if you have a lot of samples to test (see Figure 3)
• specific species with a reasonable level of accuracy without the need for additional testing
• resistant strains/species.

Whilst expensive, this relatively new media type saves a lot of time in laboratories that deal with large numbers of routine urine samples.

Figure 3 Examples of a chromogenic test plate.

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This culture medium (CHROMagar^TM Orientation) is specifically designed for urine samples. The main pathogens causing UTI can be recognised by colour, so this reduces the need for confirmatory testing. Different species produce a characteristic colony colour. A: Dark pink to reddish colonies of E. coli (bottom, left) and metallic blue colonies typical of Klebsiella, Enterobacter and Citrobacter. B: Two mixed urine cultures.

Figure 3 Examples of a chromogenic test plate.

3.3 Advanced testing

Some testing methods are beyond the scope or resources of the hospital laboratory and are more likely to be found in public health and reference laboratories. MALDI-TOF and automated systems are two methods found increasingly in larger clinical laboratories. Most molecular methods (e.g. whole genome sequencing, 16S rDNA PCR, molecular typing) are currently only used widely for research and in reference laboratories. We describe some of these methods below.

Automated systems, such as Vitek II (Biomerieux), BD Phoenix (Beckton Dickinson) and Beckman-Coulter-MicroScan are used in some clinical laboratories. Effectively these are an automated version of the biochemical test strips, with more reagents and analytic software. The cost of such systems is high, but less work is needed to complete the tests. A further advantage is that AST can be done at the same time.

4 Identifying the key pathogens in global AMR surveillance

4.1 The thirteen key pathogens

Thirteen key bacterial pathogens in humans have been identified by the World Health Organization (WHO) as the focus of the GLASS surveillance programme for AMR (Table 3) (WHO, 2023). These are all pathogens that a hospital laboratory would normally be able to identify from one or more of four specimen types.

All of the GLASS target pathogens are listed on the WHO’s priority pathogen list (WHO, 2024). E. coli, K. pneumoniae and Acinetobacter all have globally widespread strains which are multi-drug resistant (MDR), even pan-resistant to antibiotics. Resistance can spread readily between the species and these organisms have proven capacity for outbreaks and spread between institutions. Such resistant strains are harder and more expensive to treat, wasting resources and posing a threat to modern medicine. For example, patients on chemotherapy need frequent antibiotics both for treatment and prophylaxis. If they encounter resistant organisms, then these patients have a high risk of dying from infection even if their cancer was curable.

Enteric pathogens like Salmonella and Shigella are of increasing AMR and public health importance. For Salmonella there are broadly two areas of concern: zoonotic infections causing diarrhoea, and sometimes invasive infection in patients with other infections such as HIV or malaria, and S. typhi and paratyphi causing enteric fever. For Shigella, these organisms are highly transmissible by the faeco-oral route, and AMR is a problem in areas with poor sanitation.

Other GLASS target pathogens affect vulnerable populations; for example, H. influenzae, which can cause severe infections in children.

Activity 10: Usual isolation site of GLASS target pathogens

For each of the images below, can you identify the site you would expect them to be isolated from? Note: for some pathogens, there may be more than one site.

Click 'reveal discussion' to see the answer.

S. aureus tends to form grape-like clusters with each bacterium being up to 1 μm in diameter.

Which site or sites in the body is S. aureus isolated from?

Discussion

S. aureus is a common cause of infection in the community and of healthcare associated infection (HCAI). Sepsis or bloodstream infection mostly starts from an infective focus such as a wound, infected IV line or osteomyelitis. S. aureus can also cause skin and soft tissue infections such as boils and abscesses. MRSA strains of S. aureus are a particular threat in healthcare settings. A significant percentage of people carry S. aureus asymptomatically in their nose and on the skin. It can lead to infection if the body’s defences are breached, for example by a wound or medical procedure.

For GLASS, S. aureus isolated from bloodstream and lower respiratory tract samples are the focus of interest.

S. pneumoniae usually forms pairs (diplococci) or occasionally short chains.

Which site or sites in the body is S. pneumoniae isolated from?

Discussion

S. pneumoniae is a common cause of pneumonia, meningitis and ear infections, as well as infections in HIV patients. It is typically isolated from bloodstream samples in severe infections. It can also be found in upper respiratory and surface swabs, sputum and CSF cultures.

GLASS surveillance focuses on bloodstream, CSF and lower respiratory tract infection isolates.

A. baumannii

Acinetobacter are gram-negative, coccobacillary-shaped bacteria. A. baumannii accounts for most Acinetobacter infections in humans and is the species of concern. However, identification to species level is technically challenging so many sites report as Acinetobacter species only.

Which site or sites in the body are Acinetobacter species isolated from?

Discussion

Acinetobacter are classic HCAI pathogens, for example affecting patients in intensive care, particularly with open wounds or on a ventilator. Acinetobacter are typically isolated from the bloodstream, wound swabs, sputum and rarely from urine.

For GLASS, bloodstream and lower respiratory tract isolates are the focus of interest.

E. coli is a typical rod-shaped, Gram-negative bacterium, about 2 µm long.

Which site or sites in the body is E. coli isolated from?

Discussion

E. coli is the most common cause of UTI and a leading cause of bloodstream infection. Some strains have specific virulence factors allowing them to infect the urinary tract (Sarowski et al., 2019). Different pathogenic strains of E. coli are a common cause of diarrhoeal disease, for example as a result of food poisoning. E. coli are also a common cause of intra-abdominal infections such as Cholecystitis.

E. coli is typically isolated from bloodstream and urine. They can also be isolated from stools, but this requires special techniques to distinguish the pathogenic strains from commensal E. coli which is prevalent in the gut.

For GLASS, isolates from bloodstream and urine are the focus of interest.

K. pneumoniae is a typical rod-shaped, Gram-negative bacterium about 2 µm long.

Which site or sites in the body is K. pneumoniae isolated from?

Discussion

K. pneumoniae is a common cause of HCAI including UTI, pneumonia, and bloodstream and wound infections.

K. pneumoniae is typically isolated from bloodstream and urine samples and GLASS focuses on these as well as lower respiratory tract specimens. It can also be found in swabs and sputum samples, but it is harder to be sure it is acting as a pathogen rather than colonising these sites.

N. gonorrhoeae forms Gram-negative diplococci, 0.6–1 μm in diameter.

Which site or sites in the body is N. gonorrhoeae isolated from?

Discussion

N. gonorrhoeae causes the STI gonorrhoea and is commonly isolated from genital samples, and occasionally bloodstream samples in cases of disseminated infection.

For GLASS, N. gonorrhoeae isolated from genital samples are the focus.

S. typhimurium is an example of Salmonella species. Salmonella are Gram-negative, rod-shaped bacteria about 1–3 µm in length.

Which site or sites in the body are Salmonella species isolated from?

Discussion

Enteric fever is caused by S. typhi/paratyphi with the other non-typhoidal strains causing diarrhoeal disease. Enteric fever strains only affect humans, but the other strains can also be transmitted via animals and animal products. This is important in the One Health context.

For GLASS, Salmonella spp. (non-typhoidal) isolated from both stool and blood samples are the focus. For Salmonella enterica serovars blood samples are the focus.

Shigella are Gram-negative, rod-shaped bacteria typically 1–3 μm in length.

Which site or sites in the body are Shigella species isolated from?

Discussion

Shigella species cause diarrhoeal disease and cause significant morbidity in young children.

For GLASS, Shigella isolated from stool samples are the focus.

H. influenzae are gram-negative, coccobacillary-shaped bacteria

Which site or sites in the body is H. influenzae isolated from?

Discussion

H. influenzae can cause many different types of infections. These infections range from mild (ear infections in children) to serious (bloodstream infections). The most common serious infections caused by H. influenzae are pneumonia, bloodstream infections and meningitis.

For GLASS, H. influenzae isolated from CSF and the lower respiratory tract are the focus.

Neisseria meningitidis are Gram-negative diplococcus that looks like a kidney beans under a microscope

Which site or sites in the body is Neisseria meningitidis isolated from?

Discussion

Neisseria meningitidis cause meningococcal disease. The two most common types of meningococcal infections are meningitis and bloodstream infections.

For GLASS, Neisseria meningitidis isolated from blood and CSF are the focus.

P. aeruginosa are gram negative rod shaped bacteria

Which site or sites in the body is P. aeurginosa isolated from?

Discussion

P. aeruginosa can cause infections in the blood, lungs, urinary tract or other parts of the body after surgery. Patients in healthcare settings are at highest risk.

For GLASS, P. aeurginosa isolated from blood and the lower respiratory tract are the focus.

Your laboratory is almost certainly identifying other organisms than the ones listed in Activity 10. However, the focus of this course is on some of the issues around identifying the eight key GLASS pathogens under global AMR surveillance. Your laboratory should have comprehensive standard operating procedures (SOPs) for isolating and identifying these organisms. SOPs are unambiguous written instructions covering routine laboratory operations as well as the organisation and management of the laboratory. In the section that follows we discuss some important considerations about the identification of different types of bacteria.

4.2 Identifying Gram-positive cocci

Your laboratory needs to be able to reliably confirm the identification of S. aureus and S. pneumoniae (see Figure 4).

Figure 4 How to identify S. aureus and S. pneumoniae.

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Simplified scheme for identifying Staphylococci and Pneumococci for surveillance purposes. Note that this does not include Enterococci.

Figure 4 How to identify S. aureus and S. pneumoniae.

Enterococci can show any of the three patterns of haemolysis. They are generally Lancefield Group D can be confirmed using biochemical tests such as aesculin hydrolysis or litmus milk reduction. They ferment a range of sugars including lactose.

For S. aureus, a presumptive identification is done by the characteristic appearance of colonies on agar and a positive catalase test. A minimum of two tests from the following must be used to confirm identification: tube coagulase, slide coagulase, or DNAse. Alternatively, MALDI-TOF can be used.

S. pneumoniae is fastidious (Figure 5). For cultures grown in conditions that should support its growth, such as on blood agar under CO₂, the organism is suspected by its typical appearance on solid media. This, plus a positive optochin sensitivity or bile solubility test, is usually sufficient to confirm species. Latex agglutination tests are also available. If using MALDI-TOF, optochin or bile solubility testing must also be performed, as MALDI-TOF is, at present, unable to distinguish between S. pneumoniae and viridans Streptococci.

Maximise

Figure 5 Appearance of S. pneumoniae colonies on blood agar.

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Note the green colour of the medium caused by alpha-haemolysis and the ‘draughtsman’ colonies caused by the older centre collapsing on itself as the colony grows outwards on the agar.

Figure 5 Appearance of S. pneumoniae colonies on blood agar.

4.3 Identifying Gram-negative organisms

Gram-negatives are found in a range of clinical samples. As they are common causes of UTI, urine culture media are designed to isolate them. They are also an important cause of bloodstream infection – either Gram-negative sepsis, for example from a urinary source, biliary sepsis or an IV line infection, or as enteric fever caused by Salmonella typhi or paratyphi so can be isolated from blood cultures. When found in superficial swabs they are often growing as contaminants or colonising an existing wound.

The approach taken to identify Gram-negative organisms depends on several factors.

1. The site of the body where the sample was taken. In Activity 10, for example, you learned which type of sample would be most likely to yield which key pathogen.
2. The likelihood of the sample from this site containing a mixture of organisms – commensals or contaminants
3. Whether there is already a presumptive identification. For example, stool samples are usually cultured on media which selects for enteric pathogens. When S. typhi and S. paratyphi are suspected, blood cultures may also be taken to look for these organisms. The identification process of these organisms from blood cultures will be longer than from stool samples as you do not have the benefit of seeing the appearance on the selective medium.

In practice, identification of Gram-negative organisms often proceeds by a process of elimination; one positive test result dictates the choice of a secondary test and so on. This process requires laboratory technologists to have the appropriate knowledge and skills to perform this screening process.

The following sections outline how the six Gram-negative key GLASS surveillance pathogens are identified for laboratories without access to MALDI-TOF/automated systems.

4.3.1 Enterobacterales species

E. coli and K. pneumoniae are two amongst a variety of coliforms (Enterobacterales).

Your laboratory is likely to be using a differential, indicator medium based on lactose fermentation – usually either CLED or MacConkey – to isolate these organisms from urine samples (Figure 6). For blood samples you will most likely be identifying all Gram negatives.

Figure 6 (A) Yellow, lactose-fermenting colonies of E. coli on CLED. (B) A non-lactose fermenter (NLF) on CLED showing no significant change to the greenish-blue colour of the medium.

Figure 7 How to identify Enterobacterales for laboratories with access to basic tests only (adapted from Yong Ng et al., 2010).

Resistant organisms of some species are more important pathogens from the Public Health point of view than others. For example, K. pneumoniae can cause big HCAI outbreaks, and there are widespread transmissible MDR uropathogenic E. coli strains (Pitout and DeVinney, 2017). Other Enterobacterales species, however, tend to be found more sporadically.

K. pneumoniae is the only Klebsiella species which is a current focus for GLASS, because it is responsible for the majority of Klebsiella infections, including drug-resistant infections. Other Klebsiella species can still cause severe infections but have not been responsible for such extensive outbreaks. In practice, identifying as far as Klebsiella is enough for clinical purposes and many laboratories will not have the facilities for identification to species level. In this case, further identification of Klebsiella isolates, for example MDR strains or from outbreaks, will have to be done at a reference laboratory.

As discussed in Section 3.2 though, only sending the resistant ones to the reference laboratory for species identification could lead to bias in the surveillance figures. This would make it hard to tell the prevalence of the resistant strains in the area accurately. However, it has the major benefit of allowing you to be aware of which resistant Klebsiella species and strains are present in your hospital.

4.3.2 Haemophilus influenzae

Identification of Haemophilus species requires a combination of methods. Colonies on blood or chocolate agar may be presumptively identified by colony morphology. Haemophilus species produce colonies that are flat, convex and grey-white on blood agar. Following presumptive identification, further techniques, including MALDI-TOF or biochemical tests can be used to further identify the species.

4.3.3 Enteric pathogens

Your laboratory will almost certainly identify a wide variety of enteric pathogens from stool samples. For GLASS, the focus is on Salmonella and Shigella.

The first step in identification is the appearance of colonies on solid media. For example, on XLD Shigella colonies are red, while Salmonella are pink with a black middle (Figure 8). Both organisms have pale colonies on MacConkey agar as they are non-lactose fermenters (NLF). They are also urease negative.

Figure 8 Salmonella colonies in a mixed culture on XLD agar.

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The black colour is caused by hydrogen sulfide production and the colony is otherwise colourless as the organism does not ferment the lactose which is present in the medium – in contrast to most non-pathogenic, enteric coliforms. The other colonies are other enteric flora, such as E. coli.

Figure 8 Salmonella colonies in a mixed culture on XLD agar.

4.3.4 Pseudomonas aeruginosa

P. aeruginosa is a glucose non-fermenting Gram-negative rod bacteria that can grow under a variety of conditions, surviving temperatures of up to 42°C.

Pseudomonas species have no specific nutritional requirements and are non-fastidious; therefore, they grow well on all standard laboratory media. They produce colonies that are usually large and smooth with flat edges. P. aeruginosa is oxidase-positive; therefore, when coupled with colonial morphology, the results of the oxidase test can aid in providing preliminary identification for isolates.

4.3.5 Acinetobacter

Acinetobacter are non-fermenting organisms which can be differentiated from other non-fermenters such as Pseudomonas species because they are oxidase negative (Figure 9). Biochemical tests like API would be needed to confirm if necessary, but phenotypic tests to identify at species level are not very reliable and identification to species level requires specialist molecular testing. MDR isolates are best confirmed at the reference lab (Vijayakumar et al., 2019).

Figure 9 Oxidase test – used to distinguish Acinetobacter from Pseudomonas species.

4.3.6 Neisseria gonorrhoeae

N. gonorrhoeae is a fastidious and delicate organism and requires careful handling. It doesn’t survive drying or temperatures much below body temperature. It is cultured on special media such as Modified New York City (MNYC) or Thayer Martin media, and grown in a CO₂ enriched atmosphere at 35–36 ºC, otherwise it will not grow at all.

Initial identification from culture media is by Gram stain (Gram-negative cocci) and oxidase test (positive). Confirmatory tests include:

• individual biochemical tests or test strips (e.g. API-NH, Biomerieux)
• immunological tests, for example a slide-based co-agglutination test of which there are many commercial kits available (e.g. Phadebact® Monoclonal GC Test, MKL diagnostics).

4.3.7 Neisseria meningitidis

Neisseria meningitidis is closely related to Neisseria gonorrhoeae but causes an entirely different disease. Like N. gonorrhoeae it is fastidious, requiring similar specialist media and growth conditions. Initial identification from culture media is by Gram stain (Gram-negative cocci) and oxidase test (positive) with follow up confirmatory biochemical, immunological and enzyme tests where MALDI-TOF is not available. At least two of these tests are required to confirm Neisseria species.

5 Fundamentals of quality control for isolating and identifying bacteria

In a clinical microbiology laboratory several factors can affect the accuracy and reliability of test results. Quality control (QC) measures are therefore put in place to monitor whether tests perform as expected and do so reliably. You will learn more about QC and other Quality procedures in the Quality assurance and AMR surveillance course.

6 Identifying pathogens in your workplace

You should now have a better understanding of the microbiology techniques used in a variety of settings for identifying bacteria, and in particular the GLASS priority pathogens. Before you finish the course, complete this final activity, which will help you think about your laboratory’s diagnostic capacity.

7 End-of-course quiz

Well done – you have reached the end of this course and can now do the quiz to test your learning.

This quiz is an opportunity for you to reflect on what you have learned rather than a test, and you can revisit it as many times as you like. 

• End-of-course quiz

Open the quiz in a new tab or window by holding down ‘Ctrl’ (or ‘Cmd’ on a Mac) when you click on the link.

8 Summary

In this course you have learned about microbiology tests used to isolate and identify key human pathogens which are the focus of the WHO GLASS programme. Some of these tests are routinely performed in a hospital microbiology laboratory while other, more advanced techniques are used only in reference laboratories. You have also been introduced to factors related to sample quality and sample handling which can negatively impact testing, and to QC measures which can help ensure the accuracy and reliability of test results.

You should now be able to:

• know where samples are obtained from and reflect on the process by which bacterial samples are processed in your workplace
• describe the principles of laboratory tests used to isolate and identify key bacterial pathogens in human health, that are the focus of the GLASS programme
• know when and why advanced testing such as mass spectrometry and automated systems are used
• know the importance of procedures designed to ensure the quality of laboratory work relating to isolating and identifying bacteria in your workplace.

Now that you have completed this course, consider the following questions:

• What is the single most important lesson that you have taken away from this course?
• How relevant is it to your work?
• Can you suggest ways in which this new knowledge can benefit your practice?

When you have reflected on these, go to your reflective blog  and note down your thoughts.

9 Your experience of this course

You’ve now reached the end of this course. If you’ve enrolled on a pathway, please go back to the pathway page and tick the box to confirm that you’ve completed this course. On the pathway page you’ll see both your progress so far as well as the other courses you need to complete in order to achieve your Certificate of Completion for that pathway.

Now that you have completed this course, take a few moments to reflect on your experience of working through it. Please complete a survey to tell us about your reflections. Your responses will allow us to gauge how useful you have found this course and how effectively you have engaged with the content. We will also use your feedback on this pathway to better inform the design of future online experiences for our learners.

Many thanks for your help.

Now go to the survey.

References

Acknowledgements

This free course was collaboratively written by Liz Sheridan and Sarah Palmer, and reviewed by Priya Khanna, Melanie Bannister-Tyrrell, Skye Badger, Claire Gordon, Natalie Moyen and Hilary MacQueen. The course was reviewed and updated by Ben Amos, Vikki Haley and Rachel McMullan in 2025.

Except for third party materials and otherwise stated (see terms and conditions), this content is made available under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 Licence.

The material acknowledged below is Proprietary and used under licence (not subject to Creative Commons Licence). Grateful acknowledgement is made to the following sources for permission to reproduce material in this free course: