In nature, bacteria usually exist as mixed-species biofilms, where they engage in a range of synergistic and antagonistic interactions that increase their resistance to environmental challenges. Biofilms are a major cause of persistent infections, and dispersal from initial foci can cause new infections at distal sites thus warranting further investigation. Studies of development and spatial interactions in mixed-species biofilms can be challenging due to difficulties in identifying the different bacterial species in situ. Here, we apply CellTrace dyes to studies of biofilm bacteria and present a novel application for multiplex labeling, allowing identification of different bacteria in mixed-species, in vitro biofilm models. Oral bacteria labeled with CellTrace dyes (far red, yellow, violet, and CFSE [green]) were used to create single- and mixed-species biofilms, which were analyzed with confocal spinning disk microscopy (CSDM). Biofilm supernatants were studied with flow cytometry (FC). Both Gram-positive and Gram-negative bacteria were well labeled and CSDM revealed biofilms with clear morphology and stable staining for up to 4 days. Analysis of CellTrace labeled cells in supernatants using FC showed differences in the biofilm dispersal between bacterial species. Multiplexing with different colored dyes allowed visualization of spatial relationships between bacteria in mixed-species biofilms and relative coverage by the different species was revealed through segmentation of the CSDM images. This novel application, thus, offers a powerful tool for studying structure and composition of mixed-species biofilms in vitro. IMPORTANCE Although most chronic infections are caused by mixed-species biofilms, much of our knowledge still comes from planktonic cultures of single bacterial species. Studies of formation and development of mixed-species biofilms are, therefore, required. This work describes a method applicable to labeling of bacteria for in vitro studies of biofilm structure and dispersal. Critically, labeled bacteria can be multiplexed for identification of different species in mixed-species biofilms using confocal spinning disk microscopy, facilitating investigation of biofilm development and spatial interactions under different environmental conditions. The study is an important step in increasing the tools available for such complex and challenging studies. IMPORTANCE Although most chronic infections are caused by mixed-species biofilms, much of our knowledge still comes from planktonic cultures of single bacterial species. Studies of formation and development of mixed-species biofilms are, therefore, required. This work describes a method applicable to labeling of bacteria for in vitro studies of biofilm structure and dispersal. Critically, labeled bacteria can be multiplexed for identification of different species in mixed-species biofilms using confocal spinning disk microscopy, facilitating investigation of biofilm development and spatial interactions under different environmental conditions. The study is an important step in increasing the tools available for such complex and challenging studies.

Actinomycosis is an infrequent bacterial infection involving Actinomyces spp and related organisms which may occur at many body sites. It can also be found in the microbiota. Actinomyces spp are described as gram-positive bacilli whereas some species grow strictly anaerobically and some facultative. Culturing is a standardized method when suspecting actinomycosis and can be a diagnostic challenge because of inhibition of microbiota and slow growth. Enriched agar plates are used when culturing fastidious bacteria and may be more selective when including antibiotics. The aim of this project was to evaluate if using antibiotic disc facilitates identification of Actinomyces spp when mixed with microbiota. A mix of microbiota was made by pooling together several species. The susceptibility of different isolates and microbiota was analysed using antibiotic discs to determine which disc to use in a trial. A trial was done by inoculating the isolates with the microbiota on agar plates, dispensing ciprofloxacin, and trimethoprim discs. A control group without antibiotic discs were also tested. Results showed variance for most isolates susceptibility. No disc performed superior in the trial, but ciprofloxacin on FAA plates incubated anaerobically gave slightly higher recovery. Both discs facilitated identification of some isolates by supressing much microbiota. Considering that the isolates had varying susceptibility it may be problematic to find one common disc. This study has given new insights on what may facilitate identification. Further studies are needed to determine if antibiotic discs could facilitate identification of Actinomyces and needs testing on clinical samples using larger sample size.

Aerococcus spp. är grampositiva bakterier som anses vara patogener hos människor och har länge varit svåra att identifiera på grund av likheten med andra bakterier. Tidigare forskning förlitade sig på 16S rRNA-sekvensering för artbestämning, men införandet av MALDI-TOF MS revolutionerade artidentifieringen. Med hjälp av MALDI-TOF MS, kombinerad med omfattande helgenomanalyser, har Choi et. al år 2023 visat att Aerococcus urinae kan delas in i flera taxonomiska grupper: Aerococcus tenax, Aerococcus mictus, Aerococcus loyalae samt Aerococcus urinae. Huvudsyfte med denna studie var att utvärdera det nya MALDI-biblioteket för dessa arter men även kartlägga deras förekomst i urin och blod i Skåne. Det möjliggjordes med 223 Aerococcus-positiva stammar från urinodlingar och 130 från blododlingar, varav totalt 276 tidigare kallad A. urinae analyserades. Metoden var uppdelad i tre huvudsakliga block: etablering av PCR som referensmetod, en förstudie där både MALDI-TOF MS och PCR kördes parallellt, och slutligen en PCR-screening för att försöka maximera upptäckten av nya Aerococcus-arter. Resultaten har visat att endast några av dessa arter identifieras med PCR, 37 av 276 stammar, och med en hög överenstämmelse med MALDI-resultat (A. mictus n=15, A. loyalae n=20, A. urinae n=2). Studien har också indikerat en potentiell skillnad i förekomsten av Aerococcus-arter mellan blod- och urin, där A. mictus dominerar i båda, följt av A. sangunicola, och A. loyalae. Sammanfattningsvis kan inte det nya MALDI-biblioteket tillförlitligt utvärderas på grund av brist på välkaraktäriserade stammar för nya arter och begränsningar av referensmetoden. Trots detta ger studien ett underlag för att bättre utvärdera MALDI-TOF MS för Aerococcus-identifikation.

There is an urgent need for a universally effective HIV-1 vaccine, but whether a vaccine will be able to protect against HIV-1 of different clades is a significant concern. IgA from HIV-1-exposed, persistently seronegative (HEPS) subjects has been shown to neutralize HIV-1 and to block epithelial HIV-1 transcytosis, and it may target novel HIV-1 epitopes. We have tested the ability of plasma and mucosal IgA purified from HEPS subjects to neutralize HIV-1 primary isolates of different viral clades and phenotypes. IgA from two groups of HEPS subjects was tested: sex workers from Nairobi, Kenya, where clades A and D predominate, and the heterosexual partners of individuals infected by clade B virus. HIV-1-infected and low-risk uninfected individuals were included as controls. IgA purified from the blood, genital tract, and saliva of most HEPS sex workers demonstrated significant crossclade HIV-1 neutralization, whereas a more clade-restricted pattern of neutralization was found in partners of clade B-infected individuals. IgA purified from HIV-1-infected individuals also mediated cross-clade neutralization, whereas IgA from uninfected controls lacked neutralizing activity. In conclusion, mucosal and plasma IgA from HEPS subjects neutralizes HIV-1 of different clades. This ability to induce HIV-1-specific systemic and mucosal IgA may be an important feature of an effective prophylactic HIV-1 vaccine.

The increased incidence of diabetes mellitus has underscored the importance of effective management strategies, particularly in preventing complications such as diabetic foot ulcers (DFU). Chronic infections associated with DFU pose significant health risks, including lower limb amputations, highlighting the urgent need for non-invasive methods to assess skin microbiota changes. This study aimed to evaluate simple methods of sampling and quantifying skin bacteria, comparing techniques such as Gram staining, DAPI staining, fluorescence in situ hybridization (FISH), and polymerase chain reaction (PCR). Furthermore, the study investigated bacterial abundance variations across different sampling sites on the foot. Skin bacteria were sampled from healthy human volunteers using tape stripping (TPS) and swabbing. Gram-staining of the samples showed that most bacteria were found on the heel of the foot, and only Gram-positive bacteria were found on the skin of healthy study participants. However, Gram-staining showed artifacts in the form of bubbles under the microscope, which interfered with bacteria counting. PCR provided results indicating the presence of Staphylococcal species on the skin of healthy feet. DAPI staining showed images of bacteria like the ones stained with Gram staining. After using FISH-probe it was found that only a few bacteria hybridized with the probe and further optimization of the protocol is required. The study evaluated various techniques for sampling and quantifying skin bacteria and compared the number of bacteria present on the foot of healthy individuals, which may be used to identify infections before they develop into more serious conditions.

Streptococcus bovis/Streptococcus equinus-komplexet (SBSEC) inkluderar sju arter och underarter av kommensala och opportunistiskt patogena bakterier som hittas i människor, domesticerade boskap och vilda djur. Komplexets höga grad av variation i fenotypiskt uttryck reflekteras i de många namnbyten och omgrupperingar som har skett de senaste hundra åren. Infektion med SBSEC är relativt ovanligt men associerat till allvarlig sjukdom såsom bakteriemi och endokardit. Vidare finns signifikant association mellan infektion med vissa underarter och koloncancer. Bruker MALDI-TOF Biotyper används klinisk för identifiering av infektionsorsakande bakterier inom Region Skåne. I dagsläget sker identifikation av SBSEC med MALDI-TOF MS med låg upplösning endast till artnivå, och med en hög grad av felidentifiering. Det finns ett behov av korrekt identifiering av underarter inom SBSEC eftersom detta har kliniska implikationer. Därmed finns ett behov av förbättrad art- och underartsbestämning med nya bibliotek som kan ersätta eller komplettera Brukers referensbibliotek. En grupp referensisolat som tidigare art- och underartbestämts med helgenomsekvensering odlade aerobt och anaerobt utgjorde ett nytt bibliotek. Brukers bibliotek och det nya biblioteket utvärderades med en art- och underartbestämd testgrupp som odlats aerobt och anaerobt (n = 51). Studien visade en signifikant förbättring (p < 0,0001) i träffsäkerhet gällande korrekt art- och underartbestämning med MALDI-TOF MS från 67% med Brukers bibliotek till 100% med nytt bibliotek.

In this study, mould growth on wood was investigated by image analysis. The studied parameters were drying and heat-treatment temperatures (20–210°C), original and resawn surface and different wood species (spruce and larch). Small specimens—some of which were inoculated with a spore suspension—were stored under humid conditions and photographed once a week. Mould growth was assessed by image analysis. In general, results found in earlier studies regarding the influence of several parameters could be confirmed. Image analysis was found to be a useful method to quantify mould growth in an objective and reproducible way. 

Why do cockroaches and rats survive in sewers and other extremely challenging conditions? The answer is that through evolution they have been equipped with antimicrobial peptides (AMPs) that protect them from microbiological insult. In the case of cockroaches and rats, the AMPs are very potent, allowing for these challenging environments [1–3]. AMPs are the most ancient and primitive arm of the human immune system and are expressed in mammals, insects, fungus, trees—virtually every multicellular organism that coexist with bacteria, including bacteria. AMPs cover the outer barriers of our body, such as epithelium and skin, enabling us to live in coexistence with what some consider a complex organ—the microbiome [4]. As we now, through the technological advancements in microbiology, start to comprehend the complexity of the microbiome, we can also appreciate the complexity of the AMP-profile. 

De grampositiva bakterierna enterokocker tillhör normalfloran i tarmen hos människor samt djur. I detta arbete studeras vancomycin-variabla enterokocker (VVE) som är vanA positiva enterokocker, känsliga för vancomycin och kan utveckla resistens in vivo. Vancomycin-resistenta enterokocker (VRE) består av två arter bakterier Entrococcus faecalis och Enterococcus faecium. VVE består av Enterococcus faecium och är svåra att detektera med odlingsbaserad diagnostik och kan därför leda till att bakterien är underdiagnostiserad, samt att den tyst kan sprida sig inom sjukhusmiljön. VVE har en förmåga att kunna övergå från vancomycin-variabla till att vara vancomycin-resistenta fenotyper. Därför undersöks i denna studie vid vilken vancomycinkoncentration (5, 4, 3, 2 mg/L) som VVE kan anrikas i buljongerna inför amplifiering med hjälp av real-time polymerase chain reaction (real-time PCR). Resultatet visade att vancomycinkoncentrationen skulle behövas sänkas till 2 mg/L, dock kan bakgrundsfloran vara för hög vid denna koncentration av antibiotika.

Activities of moulds from domestic dwellings are normally classified into three groupsprimary, secondary, and tertiary colonizersaccording to the minimum relative humidity they require to colonize a substrate. With the help of isothermal calorimetry it is possible to directly measure the thermal activity from moulds as a function of climatic parameters. This makes it possible to provide more precise and detailed information of the growth behavior of these types of moulds under different temperature and relative humidity level than traditional methods. From this study, it is found that the optimal relative humidities and the recovery from drying are different for these three different colonizers. The fungal activities during desorption process are higher than during adsorption processes under the same relative humidity level for all of the samples. Such information makes it possible to model mould behavior indoors and can be used to access the risk for mould growth in the buildings.

The influence of temperature on the growth of the mould Penicillium roqueforti growing on malt extract agar was studied by correlating the produced heat (measured by isothermal calorimetry), ergosterol content (quantified by GC-MS/MS) and biomass of the mould at 10, 15, 20, 25 and 30 degrees C. The results were analysed with a simple metabolic model from which the metabolic efficiency was calculated. The results show that the impact of temperature on growth rate and metabolic efficiency are different: although the mould fungus had the highest growth rate (in terms of thermal power, which was continuously measured) at 25 degrees C, the substrate carbon conversion efficiency (biomass production divided by substrate consumption, both counted as moles carbon) was the highest at 20 degrees C. The temperature of the most rapid growth did not therefore equal the temperature of the most efficient growth.Similar articles

Two methods of quantifying fungal activity have been compared and correlated: isothermal calorimetry for measuring heat production and gas chromatography–tandem mass spectrometry (GC–MS/MS) for measuring ergosterol, a proxy for biomass. The measurements were made on four different fungi: Penicillium roqueforti, Cladosporium cladosporioides, Neopetromyces muricatus and the dry rot fungus Serpula lacrymans. The results showed linear correlations between ergosterol production and total heat production for these four fungal species during the initial fast growing stage. At the later stages heat was produced but ergosterol amount was constant. The heat produced per ergosterol amount varied from species to species and between different temperatures. This might be due to the different metabolic efficiencies of different species or the same species at different temperatures. Isothermal calorimetry can be used in fungal studies on its own or in combination with other techniques for a more complete understanding of fungal physiology.

Limosilactobacillus reuteri är en gram-positiv, heterofermentativ och fakultativt anaerob bakterie. Den tillhör Lactobacillus genus och är en del av floran i mag-tarmkanalen. L. reuteri tillhör mjölksyrabakterier och har 2-centrala metabolavägar som följermbden-Meyerhof-vägen och fosfoketolasvägen. Limosilactobacillus reuteri har initialt en hög Embden-Meyerhof aktivitet men uttrycker en mycket aktiv fosfoketolasväg under en jäsningsprocess. Syftet med detta projekt var att undersöka effekten av olika organiska föreningar som ingår i de två vägarna på flödet av laktatdehydrogenas. Enzymet har en viktig roll i produktionen av laktat. Undersökningen påbörjades med bakteriell fermentering i MRS-buljong. Bakterierna skördades vid den sena exponentiella fasen, lyserades och blandades med organiska föreningar i trietanolamin buffert. Enzymreaktionen analyserades spektrofotometriskt. Michaelis-Menten-principer tillämpades för att erhålla disassociationskonstanten för varje reaktion. Resultaten visar signifikant inhibering av Adenosindifosfat, nikotinamidadenindinukleotid och xylulosa-5-fosfat. Slutsatsen av studien är att reglering av laktatdehydrogenas aktivitet följer en annan mekanism jämfört med Lactococcus lactis. Denna studie kräver ytterligare optimering med varierande koncentrationer av reducerad nikotinamidadenindinukleotid för att observera enzymregleringen under dynamiska förhållanden. Det skulle även gynna undersökningen att hitta potentiella aktivatorer för enzymet.

Streptococcus bovis/Streptococcus equinus-komplexet (SBSEC) är en grupp avgrampositiva bakterier som hör till gruppen D-streptokocker och som normaltfinns i mag- och tarmkanalen hos både människor och djur. Dessa bakterier är oftaassocierade med bakteriemi, endokardit och kolorektalcancer. Matrix-assistedlaser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)har revolutionerat mikrobiologisk diagnostik genom snabb och exakt identifieringav mikroorganismer, men begränsas av dess förmåga att skilja nära besläktadearter och underarter. Det finns ett behov av korrekt identifiering av underarterinom SBSEC eftersom detta har kliniska implikationer. Därför finns det behov attvalidera det nya MALDI-biblioteket men även att undersöka om det finns unikabiomarkörer för arter och underarter inom SBSEC. Totalt 184 isolat av SBSEC,identifierade till art- och underartsnivå genom helgenomsekvensering, odlades påblodagar under aeroba förhållanden och FAA-agar under anaeroba förhållanden. För preparering inför analys användes tre metoder: direktöverföring, utökaddirektöverföring, samt extraktion i rör. Genom att analysera masspektra insamladefrån en initial testgrupp (n=111), undersöktes möjligheten att identifiera unikabiomarkörer för varje art och underart. Studien visade imponerande noggrannhet iatt exakt identifiera både arter och underarter, där 98% av de testade stammarnakorrekt klassificerades till rätt art och underart med alla tre använda preparationsmetoderna, samt lyckades identifiera unika biomarkörer för SBSEC. Slutsatsen är att det ny utvecklade MALDI-biblioteket har en hög precision ocheffektivitet i art- och underartsidentifieringen av SBSEC-isolat. Identifieringen avunika biomarkörer för specifika arter och underarter inom SBSEC var möjlig menbehöver valideras för att säkerställa tillräckligt unika och stabila biomarkörer somkonsekvent kan skilja mellan olika arter och underarter.

Denna studie är en läromedelsanalys som är kliniskt mikrobiologiskt inriktad. Syftet var att undersöka hur klinisk mikrobiologi beskrivs i läromedel efter läroplanen för grundskolan (Lgr 22), i jämförelse med läromedel som är skrivna före ( Lgr 11). Samtidigt undersöktes om alla läromedel täcker det som (Lgr 22) kräver mikrobiologiskt. Läromedlen har även jämförts innehållsmässigt med hjälp av en kvantitativ samt kvalitativ innehållsanalys. Den kvantitativa analysen redovisas i två tabeller ( Tabell 1 och Tabell 2). I tabellerna redovisas i vilka kapitel klinisk mikrobiologi framträder samt begrepp/ fenomen som förekommer i de olika läromedlen. Den kvalitativa analysen är en innehållsanalys där fenomen/begrepp/ biologiska processer i de olika läromedlen analyseras på en djupgående nivå. Studien omfattade en analys av fyra läromedel, två digitala samt två trycka. Ett av läromedlen är utformad 2015 (Gleerups i trycktform), medan de andra tre är skrivna 2022 (Gleerups, Liber och NE). Resultatet som åstadkoms visar att alla läromedel täckte kraven för mikrobiologi i LGR 22. Studien visar att synen på klinisk mikrobiologi inte har ändrats efter pandemin, vilket kunde observeras vid jämförelse av böckerna som skrevs 2022, med boken från 2015.Däremot finns en signifikant skillnad mellan Gleerups (2015) samt Gleerups (2022) läromedlet.

The prevailing form of bacterial infection is within the urinary tract, encompassing a wide array of bacteria that harness the urinary metabolome for their growth. Through their metabolic actions, the chemical composition of the growth medium undergoes modifications as the bacteria metabolize urine compounds, leading to the subsequent release of metabolites. These changes can indirectly indicate the existence and proliferation of bacterial organisms. Here, we investigate the use of an electronic tongue, a powerful analytical instrument based on a combination of non-selective chemical sensors with a partial specificity for data gathering combined with principal component analysis, to distinguish between infected and non-infected artificial urine samples. Three prevalent bacteria found in urinary tract infections were investigated, Escherichia coli, Klebsiella pneumoniae, and Enterococcus faecalis. Furthermore, the electronic tongue analysis was supplemented with 1H NMR spectroscopy and flow cytometry. Bacteria-specific changes in compound consumption allowed for a qualitative differentiation between artificial urine medium and bacterial growth.

Avhandlingen undersöker orala bakteriers svar till utifrån kommande signaler på en molekylärbiologisk nivå. I delarbete I kartläggs proteiner med serin/treonin/ tyrosin fosforylering i relation till det generella proteomet hos en oral streptokock (Streptococcus gordonii DL1). De identifierade fosfoproteinerna kunde kopplas till olika bakteriella processer, varav flera av intresse ur kariessynpunkt. Jämförelse mot andra bakteriers fosfoproteom visade många likheter, vilket är av intresse för identifiering av delade fosforyleringsprofiler. 

I delarbete II undersöks skillnader i det generella proteomet hos S. gordonii DL1 mellan tillväxtfaserna planktonisk och biofilm, samt reglerande effekter av salivmucinet MUC5B på biofilmscellerna proteinuttryck. Skillnaderna i proteinuttryck mellan tillväxtfaser ger ledtrådar om bakteriernas mekanismer för anpassning till biofilmstillväxt. 

Delarbete III studeras salivmucinet MUC5Bs reglerande roll på vidhäftning och metabolism i biofilmer med kliniska isolat av S. gordonii CW samt Actinomyces naeslundii CW. S. gordonii ökade vidhäftningen av A. naeslundii till MUC5B. Båda arterna kunde också använda MUC5B som enda näringskälla under tidig biofilmsbildning, både enskilt och tillsammans. Responserna som MUC5B framkallade i biofilmerna (paper II och III) verkar främja kolonisering av kommensaler och samtidigt nedreglera kariesrelaterade aktiviteter. 

Mikrobiologiska studier fokuserar ofta på dysbiosis och sjukdomsutveckling, men mekanismer som bibehåller eubiosis är minst lika viktiga för att förstå hur oral hälsa kan främjas. Resultat kopplade till orala bakteriers svar på utifrån kommande signaler kan bidra till framtida utveckling av nya strategier för prevention och identifiering av prediktiva biomarkörer för oral hälsa. 

Biofilm-associated infections, which are able to resist antibiotics, pose a significant challenge in clinical treatments. Such infections have been linked to various medical conditions, including chronic wounds and implant-associated infections, making them a major public-health concern. Early-detection of biofilm formation offers significant advantages in mitigating adverse effects caused by biofilms. In this work, we aim to explore the feasibility of employing a novel wireless sensor for tracking both early-stage and matured-biofilms formed by the medically relevant bacteria Staphylococcus aureus and Pseudomonas aeruginosa. The sensor utilizes electrochemical reduction of an AgCl layer bridging two silver legs made by inkjet-printing, forming a part of near-field-communication tag antenna. The antenna is interfaced with a carbon cloth designed to promote the growth of microorganisms, thereby serving as an electron source for reduction of the resistive AgCl into a highly-conductive Ag bridge. The AgCl-Ag transformation significantly alters the impedance of the antenna, facilitating wireless identification of an endpoint caused by microbial growth. To the best of our knowledge, this study for the first time presents the evidence showcasing that electrons released through the actions of bacteria can be harnessed to convert AgCl to Ag, thus enabling the wireless, battery-less, and chip-less early-detection of biofilm formation.

Since the progression of biofilm formation is related to the success of infection treatment, detecting microbial biofilms is of great interest. Biofilms of Gram-positive Staphylococcus aureus and Streptococcus gordonii bacteria, Gram-negative Pseudomonas aeruginosa and Escherichia coli bacteria, and Candida albicans yeast were examined using potentiometric, amperometric, and wireless readout modes in this study. As a biofilm formed, the open circuit potential (OCP) of biofilm hosting electrode (bioanode) became increasingly negative. Depending on the microorganism, the OCP ranged from −70 to −250 mV. The co-culture generated the most negative OCP (−300 mV vs Ag/AgCl), while the single-species biofilm formed by E. coli developed the least negative (−70 mV). The OCP of a fungal biofilm formed by C. albicans was −100 mV. The difference in electrode currents generated by biofilms was more pronounced. The current density of the S. aureus biofilm was 0.9‧10−7 A cm−2, while the value of the P. aeruginosa biofilm was 1.3‧10−6 A cm−2. Importantly, a biofilm formed by a co-culture of S. aureus and P. aeruginosa had a slightly higher negative OCP value and current density than the most electrogenic P. aeruginosa single-species biofilm. We present evidence that bacteria can share redox mediators found in multi-species biofilms. This synergy, enabling higher current and OCP values of multi-species biofilm hosting electrodes, could be beneficial for electrochemical detection of infectious biofilms in clinics. We demonstrate that the electrogenic biofilm can provide basis to construct novel wireless, chip-free, and battery-free biofilm detection method.

This paper presents results from dynamic calorespirometric measurements on the two mould fungi Penicillium roqueforti and P. camemberti growing on agar. The measurements were made with two isothermal heat conduction calorimeters connected by a tube. In one of the calorimeters, the sample was placed and the other contained a carbon dioxide absorbent. Pressure sensors were connected to both the ampoules. The equipment also contained a valve on the tube that was opened and closed at regular intervals. Measurements were started at normal atmospheric pressure and gas composition, and continued after oxygen was consumed. The response of the fungi to the changing gas composition was followed and gas exchange ratios and metabolic enthalpies were calculated by approximate methods.