Assessment and verification of commercially available pressure cookers for laboratory sterilization

Microbiology laboratory experiments require careful maintenance of sterile reagents and tools as well as the sterilization of waste prior to disposal. However, steam autoclaves typically used for this purpose may not be readily accessible to everyone in the scientific community, such as K-12 teachers, researchers in the field, or students in under-funded laboratories. This work examines the use of commercial electric pressure cookers as an alternative method for the sterilization of media, instruments, and waste. Four commonly available brands of pressure cooker were tested for their ability to sterilize microbiological media, a variety of metal instruments, and high-titer microbial cultures. All four pressure cookers were able to sterilize these starting materials as well as a range of microbial types, including Gram-positive bacteria, Gram-negative bacteria, filamentous fungi, unicellular fungi, and mixed environmental samples. Only the Instant Pot, however, was able to sterilize autoclave tester ampoules of Geobacillus stearothermophilus spores. These results suggest that, depending on the nature of the work undertaken, store-bought pressure cookers can be an appropriate substitute for commercial autoclaves. Their adoption may also help increase the accessibility of science to a broader range of investigators

The hetZ Gene Regulates Heterocyst Formation in Anabaena sp. strain PCC 7120

To form a complex multicellular organism, stem cells must differentiate into each cell/tissue type along proper spatiotemporal scales. The study of differentiation and organismal development has historically been conducted in prokaryotes due to their genetic and morphological simplicity. Anabaena sp. strain PCC 7120 is a multicellular filamentous cyanobacterium that differentiates a morphologically distinct secondary cell type, the heterocyst, in response to a lack of combined environmental nitrogen. Heterocysts are regularly spaced along filaments and fix atmospheric dinitrogen to maintain organismal viability in its absence. Previous work suggested that the hetZ gene is involved in heterocyst differentiation, but the insertional mutants created produced inconsistent phenotypes, so a specific role was not assigned. In this work, a clean hetZ mutant incapable of heterocyst differentiation was generated and the mutation was complemented with the reintroduction of hetZ alone. Overexpression of hetZ bypassed a mutation of hetR, the master regulator of heterocyst differentiation that controls biological pattern formation, but not a mutation of hetP, a regulator of commitment to a differentiated cell fate, which places hetZ roughly between these processes. A protein-protein interaction study showed that HetZ interacts with both HetR and itself. Assessment of transcriptional fusions between the hetZ, hetR, hetP, and patS (an inhibitor of HetR) promoter regions and GFP, and overexpression of HetR in a hetZ mutant resulted in the differentiation of heterocyst-like cells, together indicated that HetZ may act in concert with HetR as an early regulator of development. Taken together, these data describe a non-linear pathway of regulation leading to heterocyst development governed by both HetR and HetZ

Identification and Characterization of Fungal Isolates from Land-applied Sewage Sludge

Approximately eight million dry tons of sewage sludge is generated in the U.S. each year, with more than half of that now land-applied as the primary method of disposal. Despite the proliferation of this practice, little is known about the microbial constituents of these noxious materials. To address this knowledge gap, we isolated and characterized fungi present in archived samples of land-disposed sewage sludge collected from the Snoqualmie National Forest (Washington State). Sludge samples were resuspended in sterile water and 15 fungal isolates were selected and purified on sabouraud dextrose agar plates supplemented with 50 mg/L of chloramphenicol. Fungal morphology was assessed and photodocumented following growth on sabouraud dextrose agar, potato dextrose agar, yeast maltose agar, and malt extract agar. Additionally, pH and temperature tolerance was assessed by growth in liquid cultures of sabouraud dextrose broth from 0 ºC to 50 ºC and determined from pH 2 to 11 at 28 ºC. Carbon source utilization was assessed using Biolog Filamentous Fungi plates. Amplification and sequencing of the ITS region, commonly used in fungal phylogenetic analysis, is in progress to identify each isolate. Phenotypic assessments of growth, carbon utilization, and lactophenol cotton blue staining revealed that sludge-associated fungi are quite varied in size and appearance, but commonly grow in a mesophilic range (10-40 ºC and pH 4-10). Interspecies variation is further evident in the percentage of 31 carbon sources utilized (63-100%). To our knowledge, this work represents the first reported assessment of the fungal community in sewage sludge wastes disposed in the Snoqualmie National Forest

Profiling Volatile Constituents of Homemade Preserved Foods Prepared in Early 1950s South Dakota (USA) Using Solid-Phase Microextraction (SPME) with Gas Chromatography–Mass Spectrometry (GC-MS) Determination

https://scholar.dsu.edu/research-symposium/1013/thumbnail.jp

Characterizing the Volatilome of Land-disposed Sewage Sludge Under Seasonal Temperature Regimes

About eight million dry tons of sewage sludge waste is generated in the US annually, with more than half of that now land-disposed on agricultural and forested lands. Though containing essential plant nutrients, sludge also harbors complex mixtures of volatile organic compounds (VOCs) that result in toxic emissions therefrom. While ecotoxic impacts to sludged ecosystems are a primary concern, the stifling emissions are most obvious to and disconcerting for the public, which has led to increasing concerns for the safety of this practice. The large-scale disposal of sludge in the temperate rainforests of the Puget Sound Watershed has resulted in acute toxicity to macrobiota, and despite escalating concerns for detrimental impacts, little is known about the total VOC inventories, or “volatilomes,” of these complex wastes. To address this knowledge gap, we characterized VOC emissions from forest-disposed sewage sludge over a range of seasonal temperature regimes. We also incubated sludge samples at the more extreme 100 o C to assess the “complete volatilome.” After 1-hr incubations in gastight vials, VOCs accumulated in the headspace were sampled with a gastight syringe and analyzed with gas chromatography-mass spectrometry to generate distinct chemical fingerprints of sludge sample volatilomes over the range of temperatures. Total integrated chromatographic peak areas increased with temperature, indicating increased VOC production. Sludge volatilomes were dominated by a multitude of aliphatics and aromatics, with comparatively lesser emissions of alcohols, esters, aldehydes, terpenes, and nitrogen-, sulfur-, and halogen-containing compounds

Profiling Volatile Emissions from Sewage Sludge Land-Applied Across Washington State (USA) Using Headspace Sampling with Gas Chromatography-Mass Spectrometry (GC-MS) Determination

https://scholar.dsu.edu/research-symposium/1012/thumbnail.jp

Assessment and verification of commercially available pressure cookers for laboratory sterilization.

Laboratory science requires careful maintenance of sterile reagents and tools as well as the sterilization of waste prior to disposal. However, steam autoclaves typically used for this purpose may not be readily accessible to everyone in the scientific community, such as K-12 teachers, researchers in the field, students in under-funded laboratories, or persons in the developing world who lack funding and resources. This work examines the use of commercial electric pressure cookers as an alternative method for the sterilization of media, instruments, and waste. Four commonly available brands of pressure cooker were tested for their ability to sterilize microbiological media, a variety of metal instruments, and high-titer microbial cultures. All four pressure cookers were able to sterilize these starting materials as well as a range of microbial types, including Gram-positive bacteria, Gram-negative bacteria, filamentous fungi, unicellular fungi, and mixed environmental samples. Only the Instant Pot, however, was able to sterilize autoclave tester ampoules of Geobacillus stearothermophilus spores. These results suggest that, depending on the nature of the work undertaken, store-bought pressure cookers can be an appropriate substitute for commercial autoclaves. Their adoption may also help increase the accessibility of science to a broader range of investigators

Profiling Volatile Constituents of Homemade Preserved Foods Prepared in Early 1950s South Dakota (USA) Using Solid-Phase Microextraction (SPME) with Gas Chromatography–Mass Spectrometry (GC-MS) Determination

An essential dimension of food tasting (i.e., flavor) is olfactory stimulation by volatile organic compounds (VOCs) emitted therefrom. Here, we developed a novel analytical method based on solid-phase microextraction (SPME) sampling in argon-filled gas sampling bags with direct gas chromatography⁻mass spectrometry (GC-MS) determination to profile the volatile constituents of 31 homemade preserves prepared in South Dakota (USA) during the period 1950⁻1953. Volatile profiles varied considerably, but generally decreased in detected compounds, complexity, and intensity over three successive 2-h SPME sampling periods. Volatile profiles were generally predominated by aldehydes, alcohols, esters, ketones, and organic acids, with terpenoids constituting much of the pickled cucumber volatiles. Bisphenol-A (BPA) was also serendipitously detected and then quantified in 29 samples, at levels ranging from 3.4 to 19.2 μg/kg, within the range of levels known to induce endocrine disruption effects. Absence of BPA in two samples was attributed to their lids lacking plastic liners. As the timing of their preparation coincides with the beginning of BPA incorporation into consumer products, these jars may be some of the first BPA-containing products in the USA. To the best of our knowledge, this is the first effort to characterize BPA in and volatile profiles of rare historical foods with SPME