Gram Staining and Investigations

1. Gram Staining
   
   • What is it?
   • How is it done?
   • Bacterial Shapes
   • Bacterial Cell Wall Components
   • Interpreting a Gram Stain
   • Converting this information into a diagnosis
2. Serology
3. Full Blood Count
4. PCR Test
5. Blood Culture
6. Quiz

• Patients presenting with an infection need accurate diagnosis of the infective organism they are infected with to begin targeted treatment.
• To achieve this, a number of investigations can be carried out to identify the exact organism and, with some tests, which drugs will treat it.
• A Gram stain is a common initial investigation used in suspected bacterial infection. It involves looking at the colour and shape of a stained bacterial sample under a microscope.
• Serology is a common investigation used to detect antigens or antibodies in the patient's blood. It can help to diagnose many infections, for example syphilis, hepatitis and HIV, but it is not reliable for every infection or every patient.
• A polymerase chain reaction (PCR) test can be used to detect DNA or RNA within body fluids. PCR assays (laboratory tests) are usually designed to detect specific organisms and are widely used for many bacterial and viral infections. Some PCR tests can provide results quickly, but cost and availability vary.
• A blood culture can be taken to grow any bacteria that may be present within the usually sterile blood of the patient. Whatever grows in the culture can then be tested for antibacterial susceptibility (which antibiotics work against it). However, results take time, so in extremely urgent situations, treatment is started without waiting for them.
• In emergencies such as sepsis or meningitis, samples such as blood (and CSF in meningitis) are usually taken, but antibiotics are started without waiting for the results. This is called empirical treatment: antibiotics aimed at the most likely causative organism before the exact organism is known.

When a patient presents with an infection, it can often be difficult to tell the exact organism they are infected with from clinical signs and history alone. Many symptoms are common among many micro-organisms and thus investigations need to be carried out to confirm a diagnosis and begin targeted treatment.

This article covers a handful of diagnostic investigations that can be requested by a doctor to help with specific diagnosis of an infection. Some of these tests can be used for diagnosing other diseases that are not infectious in cause, but this is not within the scope of this article. Details of the methods of carrying out these investigations are not required past this level for medical students, but accurate interpretation of their results is vital.

Gram Staining

Used for: Bacterial infections

Sample collected via: Sputum sample (coughed-up phlegm), stool sample, urine sample, cerebrospinal fluid (CSF) sample, and blood (once bacteria have grown in a blood culture bottle)

What is it?

A Gram stain involves staining a sample with dyes (crystal violet and safranin), then looking at the shape and staining of the bacteria under a microscope.

Gram staining differentiates bacteria by the chemical and physical properties of their cell walls, mainly the thickness of the peptidoglycan layer (a mesh-like molecule that strengthens the bacterial cell wall). Gram-positive bacteria have a thick peptidoglycan layer, and Gram-negative bacteria have a thin one.

To understand Gram staining, an understanding of the different shapes and components of the cell walls of bacteria is required.

The Gram stain is almost always the first step in the preliminary identification of a bacterial organism. While Gram staining is a valuable diagnostic tool in both clinical and research settings, not all bacteria can be definitively classified by this technique.

How is it done?

There are 4 basic steps to performing a Gram stain:

1. A primary stain of crystal violet is applied to a heat-fixed smear of a bacterial culture (a thin film of bacteria spread on a glass slide and briefly heated so that it sticks). Both Gram-positive and Gram-negative bacteria initially take up the stain.
2. Gram's iodine (a solution of iodine and potassium iodide) is added. It acts as a mordant (a fixing agent): the iodide combines with the crystal violet to form a larger crystal violet-iodine complex that does not dissolve in water, so it is harder to wash out of the cells.
3. Rapid decolourisation with ethanol or acetone. This washes the crystal violet-iodine complex out of Gram-negative cells, because their thin peptidoglycan layer and outer membrane (an extra outer layer, see below) are disrupted by the decolouriser. Gram-positive cells have a thick peptidoglycan wall that resists decolourisation, so they stay purple.
4. The Gram-negative cells lost the crystal violet during decolourisation, so they are now colourless and cannot be seen. The sample is now counter-stained with safranin, a pink dye, to make these cells visible. Gram-positive cells stay purple and Gram-negative cells appear pink.
   

Bacterial Shapes

Bacterial shapes can be either:

• Coccus shaped (pl. cocci) - these bacteria look circular 
• Bacillus shaped (pl. bacilli) - these bacteria look like long tubes and are sometimes referred to as “rod” bacteria
• Spirillum or spiral shaped (pl. spirilla) - seen in a small number of bacteria and a relatively uncommon finding on a Gram stain

Image: The 3 shapes of bacteria. Note that cocci can arrange into singular cocci, diplococci (pairs of cocci) and larger groups such as chains and clusters

Creative commons source by CKRobinson [CC BY-SA 4.0 (https://creativecommons.org/licenses/by-sa/4.0)]

The arrangement of the bacteria within space is also important. For example:

• Streptococcus species are commonly arranged in chains of cocci, which look like strips (memory aid: STREPS are STRIPS)
• Staphylococcus species are usually arranged in clusters (similar to bunches of grapes)

This information can be very useful in distinguishing between different species that may appear similar on a Gram stain.

Figure: Gram Stain of a Streptococcus species. Notice the long chains of cocci ("strips") and purple colouring as all streptococci are Gram-positive. "Streps are strips!"

Public Domain Image [Public Health Image Library]

Figure: Gram Stain of a Staphylococcus species. Notice the clusters of cocci.

Creative commons source by Y Tambe [CC BY-SA 3.0 (http://creativecommons.org/licenses/by-sa/3.0/)]

Bacterial Cell Wall Components

Bacterial cell wall components often define how they infect the host, as well as their virulence and potential to cause disease. The cell wall components are also key in how an organism Gram stains.

There are two main categories of bacteria:

• Gram-positive
• Gram-negative

Bacterial cell wall compositions vary based upon the category of bacteria:

• Gram-positive bacteria have a plasma membrane surrounded by a THICK peptidoglycan layer
• Gram-negative bacteria have a plasma membrane, a THIN peptidoglycan layer and then an extra outer membrane. The outer layer of this membrane contains lipopolysaccharide (LPS), also called endotoxin. LPS is a virulence factor (a feature that helps bacteria cause disease): it triggers inflammation and is a major mediator (driver) of sepsis, which is one reason Gram-negative infections can be severe.

Diagram: A Gram-positive bacterial cell wall with its main components

SimpleMed original by Dr. Marcus Judge

Diagram: A Gram-negative bacterial cell wall with its main components. Note the outer membrane containing LPS, a virulence factor that can contribute to serious disease

SimpleMed original by Dr. Marcus Judge

Interpreting a Gram Stain

To reiterate; interpretation is carried out by analysing the colour and shape of the bacteria on the microscope slide.

Colour -

A thick peptidoglycan layer keeps the crystal violet stain (as a crystal violet-iodine complex) during decolourisation. As such:

• A Gram-positive bacterium is PURPLE/VIOLET on a Gram stain as its thick peptidoglycan layer keeps the crystal violet.
• A Gram-negative bacterium is PINK on a Gram stain, as its thin peptidoglycan layer loses the crystal violet and it takes up the pink safranin counterstain.

Shape -

As previously discussed, the shape of a bacterium can be either cocci, bacillus or spiral.

Figure: Gram Positive vs Gram Negative Gram stain

Created by Dr. Marcus Judge for SimpleMed using Public Domain resources from: https://pixnio.com/science/microscopy-images/staphylococcus-aureus/photomicrograph-of-spherical-cocci-gram-positive-staphylococcus-aureus-bacteria-magnified-250x# and http://www.publicdomainfiles.com/show_file.php?id=13513615212956

Converting this information into a diagnosis

Upon knowing the colour and shape of a bacterium the current differentials can be supported or rejected. This is why knowing the shape and Gram stain result of common bacteria is important for a doctor.
Here are some useful examples:

Gram positive cocci - (Purple circles)

• Staphylococcus species e.g. Staphylococcus aureus (causes cellulitis and skin abscesses)
• Streptococcus species e.g. Streptococcus pneumoniae (usually a diplococcus, i.e. in pairs). S. pneumoniae is a leading cause of community-acquired pneumonia (pneumonia caught outside hospital), middle ear infections (otitis media) and bacterial meningitis.

Often treated with: Penicillins, for example flucloxacillin for cellulitis and amoxicillin for community-acquired pneumonia. If MRSA (meticillin-resistant Staphylococcus aureus) is suspected, another antibiotic such as vancomycin is added.

Gram positive bacilli - (Purple rods)

• Clostridioides difficile, previously called Clostridium difficile (also referred to as C. diff). It is a common hospital-acquired infection that causes diarrhoea, often after a course of antibiotics. It forms spores (tough, dormant forms of the bacterium), which make it hard to eradicate.
  
• Bacillus anthracis (the cause of anthrax, a notifiable disease, i.e. doctors must report any suspected case to the UK Health Security Agency, UKHSA, which replaced Public Health England)
• Listeria monocytogenes (causes listeriosis, which is not usually serious but can cause serious or life-threatening illness in pregnancy, in newborn babies, in older people and in people with a weakened immune system)

Treatment depends on the organism. For example, C. diff is treated with oral vancomycin, and other antibiotics are stopped unless essential. Listeria meningitis is treated with intravenous amoxicillin or ampicillin (both penicillins).

Gram negative cocci - (pink circles)

• Neisseria meningitidis (causes meningococcal disease, which includes meningococcal meningitis and meningococcal septicaemia, an infection of the blood)
• Neisseria gonorrhoeae (causes gonorrhoea)

Often treated with: Cephalosporins (a large class of antibiotics, for example ceftriaxone, according to current UK guidance)

Gram-negative bacilli - (pink rods)

• Escherichia coli (the most common cause of urinary tract infections; some strains, such as E. coli O157, cause gastroenteritis)
• Salmonella typhi (causes typhoid fever)

Often treated with: Cephalosporins, adjusted once sensitivity results show which antibiotics work. Fluoroquinolones (a class of antibiotics that includes ciprofloxacin) must now only be used when other commonly recommended antibiotics are inappropriate, because they can cause disabling and potentially long-lasting side effects.

Other Investigative Techniques:

Serology

Definition: Serology refers to the study of serum (blood plasma without clotting factors) but in this context is used specifically to refer to diagnostic techniques within this field of study.

Used for: Many (but not all) infections, and some non-infective conditions, e.g. rheumatological (joint and autoimmune) conditions.

Used to detect: Exposure to or infection with organisms such as Treponema pallidum (syphilis), hepatitis viruses and HIV

Sample collected via: Blood (usually), other bodily fluids

Serological tests rely on the formation of antibodies in response to an infection (against a given microorganism). Presence of these antibodies in the blood implies that the body has begun to mount an immune response (which may be ineffective) against the organism. Results can be falsely negative early in an infection, before enough antibodies have formed, and in immunocompromised people (those with a weakened immune system), who may not make enough antibodies. Serology is therefore less reliable in these patients, and tests that detect the organism itself (such as antigen tests or PCR) may be needed.

There are several serology techniques that can be used depending on the antibodies being studied. These include: enzyme-linked immunosorbent assay (ELISA), agglutination, precipitation, complement-fixation, fluorescent antibodies and chemiluminescence to name but a few examples (detailed knowledge of these tests is not required for this topic).

These techniques often use complementary antibodies (laboratory-made antibodies that bind to the patient’s antibodies) to detect disease marker antibodies in the patient’s serum. The complementary antibodies may carry a fluorescent marker, or trigger a visible enzyme reaction, to signal a positive test.

Full Blood Count

Definition: A very common test carried out on the patient’s blood to detect the levels and characteristics of certain benchmark cells within the blood. Abnormal numbers of red blood cells, white blood cells or platelets, or abnormal red cell size, can point to a disease. Inflammatory markers (blood proteins that rise with inflammation), such as C-reactive protein (CRP), are not part of the full blood count: they are measured in a separate test. This investigation is not inherently diagnostic but can often show if a patient is infected and what cells are being affected within the blood.

Used for: All types of infections

Used to detect:

• Sepsis (the neutrophil count may be very high or very low). The full blood count is one of several blood tests taken in suspected sepsis, alongside CRP and blood cultures.
• HIV infection may be suspected (for example from a low lymphocyte count), but it is diagnosed with a specific HIV test. A separate CD4 count (CD4 T-cells are the white cells that HIV destroys) then shows how advanced the infection is.
• Epstein-Barr virus (EBV), the most common cause of glandular fever. The full blood count shows raised lymphocytes, often with atypical lymphocytes (unusual-looking, activated lymphocytes).
• Other acute viral or bacterial infections (changes in the white cell count; raised inflammatory markers such as CRP show up on the separate CRP test)

Sample collected via: Blood

A full blood count is almost always requested when a patient comes in acutely ill. It provides information such as:

• White cell count
  • Neutrophil count: Raised levels may indicate bacterial infection. They may also be raised in acute viral infections.
  • Lymphocyte count: Higher with some viral infections such as Epstein-Barr virus. EBV infects B-cells (the white cells that make antibodies), and much of the rise in lymphocytes is due to T-cells (CD8 T-cells) multiplying to attack these infected B-cells. Counts may be decreased by HIV infection, as this causes the destruction of T-cells.
  • Raised Monocytes: May be raised in bacterial infection, tuberculosis and malaria.
  • Eosinophils: Increased in some parasitic infections, especially worm (helminth) infections, for example in returning travellers and migrants.
• Red cell count - may be reduced in malaria and other parasitic infections that infect red blood cells and can cause anaemia.
• Platelet count - a reduced platelet count (thrombocytopenia) can be seen in severe sepsis.

See our dedicated article in the haematology section 

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