Acinetobacter is officially classified as a Gram-negative bacterium, but it is one of the most frequently misread organisms on a Gram stain. The cells resist the decolorization step of the staining process, which means they often retain the crystal violet dye and appear purple instead of the expected pink. Depending on the culture’s age and growing conditions, the same slide can show a confusing mix of pink, violet, and intermediate-colored cells. This Gram-variable behavior has led to delayed or incorrect treatment in clinical settings, making Acinetobacter a textbook example of why a single Gram stain result should never be taken at face value.
What Acinetobacter Looks Like on a Slide
Under the microscope, Acinetobacter cells are short, plump, rod-shaped bacteria that tend toward a rounded or coccobacillary form. They were first identified in 1911, and early descriptions already noted their stubby shape and tendency to form pairs or short chains.1Academic Press. Microbiology of Waterborne Diseases Unlike the classic long, thin rods you might picture when someone says “Gram-negative bacillus,” Acinetobacter looks more like a plump oval. In clinical lab descriptions, the term “Gram-negative coccobacilli” comes up repeatedly, and laboratory inclusion criteria for studying Acinetobacter isolates typically list them as nonlactose-fermenting, oxidase-negative, nonmotile, Gram-negative coccobacilli.2PubMed Central. Role of Matrix-Assisted Laser Desorption Ionization–Time-of-Flight Mass Spectrometry for Species Identification of Acinetobacter Strains
That rounded shape is part of the identification problem. When a microbiologist sees plump, paired organisms that stain purple on a Gram stain, the natural instinct is to call them Gram-positive cocci, not Gram-negative rods. The shape itself primes the reader to expect a staphylococcus or streptococcus, and the staining pattern can reinforce that first impression rather than correct it.
Why It Stains the Wrong Color
The Gram stain works by exploiting differences in bacterial cell walls. Gram-positive bacteria have a thick outer layer of peptidoglycan that traps crystal violet dye, so they stay purple after the alcohol wash. Gram-negative bacteria have a thinner peptidoglycan layer and an additional outer membrane; the alcohol dissolves the outer membrane, the thin peptidoglycan cannot hold the dye, and the cell picks up the pink counterstain instead. Acinetobacter has a Gram-negative cell envelope, but its wall does not always behave like one during the staining procedure.
Part of the explanation lies in the organism’s unusually robust peptidoglycan. Research on Acinetobacter baumannii has shown that the peptidoglycan layer provides significant structural rigidity to the cell, to the point where the bacteria can actually survive without the lipooligosaccharide (LOS) that normally coats the outer membrane of Gram-negative organisms. When LOS is absent, the peptidoglycan synthesized by the cell’s elongasome machinery becomes essential for maintaining cell shape and integrity.3PubMed Central. Acinetobacter baumannii Can Survive with an Outer Membrane Lacking Lipooligosaccharide Due to Structural Support from Elongasome Peptidoglycan Synthesis A thicker-than-typical peptidoglycan layer, or one that is more tightly cross-linked, could explain why the crystal violet dye is harder to wash out during the decolorization step. The cells simply hang onto it more stubbornly than other Gram-negative bacteria do.
The outer membrane also plays a role. Acinetobacter species often produce a capsule, an additional polysaccharide layer outside the outer membrane.4Academic Press. Microbiology of Waterborne Diseases This capsule can interfere with how solvents and dyes interact with the cell, potentially slowing decolorization further. The combined effect is a cell that technically has a Gram-negative architecture but does not consistently behave like one in the staining protocol.
The Growth Phase Problem
The single biggest factor that determines whether Acinetobacter stains pink or purple on a given slide is how old the culture is. When the bacteria are actively dividing during the logarithmic (log) growth phase, they tend to take the shape of short Gram-negative rods and stain pink as expected. But as the culture ages and enters the stationary phase, the cells become more coccoid, rounder and smaller, and increasingly difficult to destain.5PubMed Central. Misinterpretation of Gram Stain from the Stationary Growth Phase of Positive Blood Cultures for Brucella and Acinetobacter Species This is when misidentification is most likely.
Researchers have tried to put numbers on this phenomenon. One study used automated image analysis to count the proportion of pink versus violet cells in stationary-phase cultures of A. baumannii. The results showed that even in the stationary phase, about two-thirds of the cells stained pink while roughly a third retained the violet dye.6PubMed. An image processing-based quantification of gram variability in Acinetobacter baumannii That means a microbiologist looking at a stationary-phase smear sees a mixed population: mostly pink cells, but with a substantial minority of purple ones scattered throughout. The mix of colors on a single slide is what earns Acinetobacter its reputation as “Gram-variable.” The reasons why individual cells within the same population take up dye differently are still not fully understood, though differences in cell wall thickness, capsule production, and local microenvironment on the slide may all contribute.
This matters in the real world because blood culture bottles, where many clinical Acinetobacter infections are first detected, often flag positive only after the bacteria have been growing for many hours. By the time the lab pulls a sample for Gram staining, the organisms may already be in stationary phase and looking nothing like textbook Gram-negative rods.
When Misidentification Changes Patient Care
A misread Gram stain is not just an academic curiosity. It can directly affect which antibiotics a patient receives in the critical early hours of an infection. Gram-positive cocci and Gram-negative rods call for fundamentally different empiric treatment regimens. If Acinetobacter is mistakenly identified as Gram-positive cocci, a clinician may start vancomycin or another Gram-positive-targeted drug rather than the broad-spectrum Gram-negative coverage the patient actually needs.
Published case reports illustrate the real consequences. In one instance involving a species called Acinetobacter ursingii, blood cultures flagged positive and the Gram stain was read as showing Gram-positive cocci, mostly in pairs. Because Acinetobacter can retain the crystal violet stain and appear as a Gram-positive organism, the initial read was entirely wrong. The authors of that report emphasized that patients with serious infections may not receive appropriate therapy if antibiotics are narrowed based on such a misleading Gram stain.7Clinical Microbiology and Infection. Acinetobacter ursingii masquerading as Gram-positive cocci
A separate series of neurosurgical meningitis cases documented delayed treatment after Acinetobacter was misidentified on the initial Gram stain. Despite the late start to correct therapy, those particular patients ultimately recovered once appropriate antibiotics were given, but the delay itself represented a period of unnecessary risk.8Neurosurgery. Antibiotic-resistant Acinetobacter Meningitis in Neurosurgical Patients In both scenarios, the Gram stain gave a misleading first impression, and correct identification came only after biochemical testing or culture results caught up.
How Labs Move Beyond the Gram Stain
Given that the Gram stain alone is unreliable for Acinetobacter, clinical labs rely on a cluster of additional tests to pin down the identification. The standard preliminary workup includes checking whether the organism is catalase-positive (it is), oxidase-negative (it is), and nonmotile (it is). That particular combination, Gram-negative coccobacilli that are catalase-positive, oxidase-negative, and nonmotile, narrows the field considerably and can be performed in any routine microbiology laboratory.9PubMed Central. Identification of genus Acinetobacter: Standardization of in-house PCR and its comparison with conventional phenotypic methods
The oxidase test is particularly useful as a quick separator. Many of the other Gram-negative organisms that Acinetobacter might be confused with, such as Pseudomonas or Neisseria species, are oxidase-positive. An oxidase-negative result on a coccobacillary Gram-negative organism strongly suggests Acinetobacter.10PubMed Central. History of the taxonomy of Acinetobacter: the emergence of hospital-adapted species of global health concern For species-level identification, which matters because different Acinetobacter species have very different clinical significance and resistance profiles, labs increasingly use mass spectrometry-based methods (MALDI-TOF) or molecular techniques like PCR. These tools bypass the ambiguity of staining entirely and identify the organism based on its protein or DNA signature.
Still, the Gram stain remains the first test performed on most positive clinical specimens because it is fast, cheap, and gives immediate preliminary guidance. The key is knowing its limitations. An experienced microbiologist who sees plump, paired organisms with equivocal staining will flag the result as possibly Acinetobacter and advise clinicians to keep empiric therapy broad until definitive identification arrives.
Antibiotic-Induced Shape Changes That Complicate Things Further
Even if a lab microbiologist is well aware of Acinetobacter’s staining quirks in normal growth conditions, antibiotics can throw another curveball. Beta-lactam antibiotics, including the carbapenems that are often used as last-resort drugs against resistant Acinetobacter infections, attack the bacterial cell wall. When A. baumannii is exposed to a lethal concentration of a carbapenem like meropenem, a subset of surviving cells undergo a dramatic morphological change: they lose their rod shape entirely, swell into large spherical forms, and shed their cell walls.11PubMed Central. Non-walled spherical Acinetobacter baumannii is an important type of persister upon β-lactam antibiotic treatment
These wall-deficient spherical cells look nothing like the typical Acinetobacter coccobacillus. Without a conventional cell wall, they would stain unpredictably on a Gram stain if they stained at all. The spherical persisters can survive the antibiotic onslaught and potentially revert to normal growth once the drug pressure is removed. For laboratory purposes, the practical implication is that a Gram stain performed on a specimen from a patient already receiving beta-lactam therapy may show bizarre, unrecognizable morphologies that do not match any expected pattern. Other antibiotic classes produce less dramatic shape changes: aminoglycosides like kanamycin and fluoroquinolones like ofloxacin cause less morphological distortion, though ofloxacin-treated cells can elongate into longer rods than usual.12PubMed Central. Non-walled spherical Acinetobacter baumannii is an important type of persister upon β-lactam antibiotic treatment
Tips for Reading an Acinetobacter Gram Stain
If you are training in clinical microbiology or just trying to understand how this organism gets missed, there are a few practical points worth keeping in mind:
- Expect mixed staining: A slide with both pink and purple coccobacilli does not necessarily mean you have a polymicrobial infection. It may be a single Acinetobacter population showing its characteristic Gram-variable behavior.
- Consider culture age: If the blood culture bottle has been incubating for a long time before the Gram stain is prepared, stationary-phase morphology is more likely. The cells will be rounder and more prone to retaining the crystal violet dye.
- Do not narrow therapy prematurely: When plump, paired organisms stain purple and look like Gram-positive cocci, resist the temptation to treat only for Gram-positive infection until confirmatory tests are complete.
- Run the oxidase test early: A quick oxidase-negative result on suspicious-looking coccobacilli can point strongly toward Acinetobacter and prompt the lab to adjust its reporting.
- Account for antibiotics: If the patient is already on beta-lactams, the organisms on the slide may have aberrant morphology that does not fit any standard description.
The Role of Automated Image Analysis
The subjectivity inherent in reading a Gram stain, particularly with tricky organisms like Acinetobacter, has prompted interest in computer-assisted reading. Deep learning models trained on large datasets of clinical Gram stain images are being developed to classify bacteria automatically. One recent effort assembled a large clinical dataset of microscopy images for training these models, with A. baumannii included among the common Gram-negative bacilli in the training set alongside organisms like Klebsiella pneumoniae and Pseudomonas aeruginosa.13Nature. A Clinical Bacterial Dataset for Deep Learning in Microbiological Rapid On-Site Evaluation
The promise of these systems is consistency. A trained algorithm does not get tripped up by the mental shortcut of “purple and round means Gram-positive.” It can be taught that plump, paired organisms with mixed staining in a blood culture bottle should raise the possibility of Acinetobacter rather than defaulting to the more common cocci. Meanwhile, as referenced earlier, image-processing pipelines have already been used in a research context to quantify the proportion of pink versus violet cells in Acinetobacter cultures, giving an objective measure of Gram variability that human eyes can only estimate.14PubMed. An image processing-based quantification of gram variability in Acinetobacter baumannii Whether automated reading becomes standard in clinical labs remains to be seen, but for organisms with notoriously ambiguous staining, the potential benefit is clear.
Other Organisms That Pull the Same Trick
Acinetobacter is not the only bacterium that confounds the Gram stain. The same paper that documented the Acinetobacter misidentification case also described Brucella species causing similar confusion: small coccobacilli that can appear Gram-variable or Gram-positive depending on the preparation.15PubMed Central. Misinterpretation of Gram Stain from the Stationary Growth Phase of Positive Blood Cultures for Brucella and Acinetobacter Species Gardnerella vaginalis is another well-known Gram-variable organism. Mycobacteria do not stain reliably with the Gram stain at all and require an acid-fast stain instead. Each of these organisms, for different structural reasons, falls outside the clean pink-or-purple binary that the Gram stain was designed to create.
The lesson these organisms collectively teach is that the Gram stain is a screening tool, not a definitive identification method. It was developed in the 1880s as a way to quickly sort bacteria into two broad groups, and for most common pathogens it works brilliantly. But biology is messier than any single staining protocol can capture. For Acinetobacter, the combination of cell wall architecture, capsule production, growth-phase-dependent morphology, and resistance to decolorization creates a perfect storm of staining ambiguity. The stain still has value as a rapid first look, but it works best when the person reading it knows exactly which organisms can break the rules.